Generation and characterization of a site-specific antibody for SIRT1 O-GlcNAcylated at serine 549

Generation and characterization of a site-specific antibody for SIRT1 O-GlcNAcylated at serine 549 Abstract O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies thousands of proteins. However, the roles and mechanisms of O-GlcNAcylation have been clarified in only a few proteins, and one of the main reasons for this is the lack of site-specific anti-O-GlcNAc antibodies. Recently, we found that SIRT1, which is an NAD+-dependent deacetylase, is O-GlcNAcylated at the serine 549 site (S549) and plays a cytoprotective role under stress. However, the mechanism underlying the roles of SIRT1 O-GlcNAcylation remains unclear. Here, we describe a site-specific antibody for SIRT1 O-GlcNAcylated at S549, named SIRT1-549-O. This antibody can be used for immunoprecipitation and western blotting assays, and it can be used to recognize the endogenous levels of both human and mouse SIRT1 O-GlcNAcylation. Therefore, this antibody not only provides an effective method to further understand the roles of SIRT1 O-GlcNAcylation but also makes it possible to discover the genetic and pharmacological factors that could regulate SIRT1 activity by modulating its O-GlcNAcylation. O-GlcNAc, oxidative stress, SIRT1, site-specific antibody Introduction Post-translational modifications (PTMs) are covalent processing events that can change the properties of a protein by proteolytic cleavage or by the addition of a modifying group to one or more amino acids. These modifying groups consist of phosphate, glycans, ubiquitin, nitroso, methyl and acetyl, etc. PTMs not only increase the functional diversity of the proteome but also determine protein activity states, localizations, interactions with other proteins, and nearly all aspects of normal cell biology and pathogenesis. As is well known, phosphorylation refers to a ubiquitous protein post-translational modification. With the development and application of large numbers of site-specific antibodies for phosphorylation, the understanding of phosphorylation has been facilitated. Therefore, site-specific antibodies are powerful tools for the study of PTMs. Similar to phosphorylation, O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is also a dynamic and ubiquitous post-translational modification that occurs on the hydroxyl groups of the serine and/or threonine residues of nuclear and cytoplasmic proteins. The attachment of GlcNAc is catalyzed by O-GlcNAc transferase (OGT), which utilizes the uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) produced by the hexosamine biosynthesis pathway from glucose as the substrate (Kreppel et al. 1997). Reversely, O-GlcNAc moieties are removed from proteins by the glycoside hydrolase O-GlcNAcase (OGA) (Dong and Hart 1994). Currently, more than 4000 proteins with O-GlcNAcylation have been found. Indeed, O-GlcNAcylation participates in almost all biological processes and is involved in epigenetic regulation, protein translation, proteasomal degradation, signal transduction, stress responses, and cellular homeostasis (Hanover 2001). Although a few decades have passed since the discovery of O-GlcNAcylation, the study of O-GlcNAcylation is still progressing slowly. One of the main reasons for this is the lack of O-GlcNAcylation-specific antibodies. Some pan-O-GlcNAc antibodies have been developed including the antibodies CTD110.6 (Comer et al. 2001), RL2 (Snow et al. 1987), 18B10.C7(#3), 9D1.E4(#10), and 1F5.D6(#14) (Teo et al. 2010). Among these antibodies, CTD110.6 and RL2 are the most widely used. However, due to a certain degree of amino acid sequence dependency, these antibodies cannot detect all proteins with O-GlcNAcylation. Additionally, a few antibodies for site-specific protein O-GlcNAcylation have been reported, and all of these antibodies comprise anti-Tau (GlcNAc S400) (Cameron et al. 2013), anti-IRS2 (GlcNAc T1155), anti-H3 (GlcNAc T32) and anti-H4 (GlcNAc S47). These antibodies make it possible to study the functions and regulatory mechanisms of site-specific protein O-GlcNAcylation. SIRT1 is a NAD+-dependent deacetylase that belongs to the class III histone deacetylases (HDACs) (Smith et al. 2000). Moreover, SIRT1 plays a vital role in the regulation of metabolism (Boutant and Canto 2014), DNA repair (Choi and Mostoslavsky 2014), genomic stability (Oberdoerffer et al. 2008), the cell cycle (Brunet et al. 2004), cell survival and apoptosis (Luo et al. 2001), cellular senescence (Langley et al. 2002), and oncogenesis (Bosch-Presegue and Vaquero 2011) by deacetylating histones and various non-histone substrates. Recently, we found that SIRT1 is O-GlcNAcylated, which directly enhances the deacetylase activity of SIRT1, and the main O-GlcNAcylation site is serine 549 (S549) (Han et al. 2017). The O-GlcNAcylation of SIRT1 is elevated during genotoxic, oxidative and metabolic stress stimuli, and this process not only increases SIRT1 deacetylase activity but also protects cells from stress-induced apoptosis (Han et al. 2017). However, whether the roles of SIRT1 O-GlcNAcylation are due to the O-GlcNAcylation at S549 still needs to be confirmed. To achieve this, a site-specific antibody for SIRT1 O-GlcNAcylated at S549 will be helpful. Here, we described the generation and characterization of a rabbit polyclonal antibody, called SIRT1-549-O, which is specific for SIRT1 O-GlcNAcylated at S549. Furthermore, the utility of this antibody for biochemical applications is demonstrated. This antibody will be an effective tool for the study of the physiological and pathological effects of SIRT1 O-GlcNAcylation. Results SIRT1-549-O antibody is specific for SIRT1 peptide that is O-GlcNAcylated at Ser549 by ELISA To generate a rabbit polyclonal antibody against O-GlcNAcylated SIRT1 at S549, we synthesized two peptides spanning the sequence around S549, i.e., the O-GlcNAcylated SIRT1 peptide (CTSPPD-(O-GlcNAc)S-SVIVTLLD) and the unglycosylated SIRT1 peptide (CTSPPDSSVIVTLLD). The antibody antigen binding activity was tested by ELISA, and the results revealed that the SIRT1-549-O antibody exhibits highly specific binding activity with a calculated EC50 of 0.5 μg/mL towards the O-GlcNAcylated SIRT1 peptide but does not bind to the unglycosylated peptide (Figure 1). The lowest O-GlcNAc SIRT1 peptide-specific signal was detected at a 12 ng/mL antibody concentration. Altogether, these results indicated that the SIRT1-549-O antibody recognized the SIRT1 peptide in a manner that was dependent on the O-GlcNAcylation of SIRT1. Fig. 1. View largeDownload slide The SIRT1-549-O antibody is specific to the SIRT1 peptide O-GlcNAcylated at Ser549. ELISA with O-GlcNAcylated and unglycosylated SIRT1 peptides demonstrated the highly specific binding activity of SIRT1-549-O for the O-GlcNAcylated SIRT1 peptide. Fig. 1. View largeDownload slide The SIRT1-549-O antibody is specific to the SIRT1 peptide O-GlcNAcylated at Ser549. ELISA with O-GlcNAcylated and unglycosylated SIRT1 peptides demonstrated the highly specific binding activity of SIRT1-549-O for the O-GlcNAcylated SIRT1 peptide. SIRT1-549-O antibody is specific to O-GlcNAcylated human SIRT1 To examine whether the SIRT1-549-O antibody specifically recognizes the O-GlcNAcylated SIRT1 protein, the cell lysates of NCI-H1299 cells treated with/without the OGA inhibitor Thiamet G (TMG) were detected by western blotting (WB). Although several bands were blotted by the SIRT1-549-O antibody, there was a band with an apparent molecular weight (MW) of 120 kDa, which is the same molecular mass as the SIRT1 protein. Additionally, the intensity of the band was markedly elevated by the TMG treatment (Figure 2A, left panel). To verify whether the blotted bands were dependent on the O-GlcNAc moiety, 500 mM free GlcNAc was included while blotting with the SIRT1-549-O antibody. The results revealed that almost all of the bands were eliminated by free GlcNAc (Figure 2A, right panel). Additionally, OGT was silenced in the NCI-H1299 cells, and the cell lysates were blotted with the SIRT1-549-O antibody. As expected, OGT silencing effectively decreased the expression of OGT and the global cellular O-GlcNAcylation levels blotted by the RL2 antibody (Figure 2B). In line with the above results, OGT silencing significantly inhibited the intensity of the band blotted by the SIRT1-549-O antibody (Figure 2B). Collectively, these results demonstrated that the SIRT1-549-O antibody recognizes proteins depending on the O-GlcNAc moiety. To verify that the detected band at the position of 120 kDa was SIRT1 O-GlcNAcylated at S549, free O-GlcNAcylated SIRT1 peptide was added when the WB assays were performed using SIRT1-549-O antibody as detailed in figure 2A. The results revealed that the free peptide eliminated the band (Figure 2C). Furthermore, the NCI-H1299 cells exhibited silenced endogenous SIRT1 while expressing wild-type SIRT1 (wtSIRT1) or the mutant SIRT1 (SIRT1S549A, the Ser 549 was mutated to Ala); these findings were established and verified as previously reported (Han et al. 2017) and used to clarify the specificity of the SIRT1-549-O antibody. The results indicated that the mutation of S549 markedly decreased the intensity of the band (Figure 2D). The residual bands should indicate the detection of the endogenous SIRT1 that is not completely silenced. Fig. 2. View largeDownload slide The SIRT1-549-O antibody is specific to the O-GlcNAcylated S549 site in human SIRT1. (A) The lysates of NCI-H1299 cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O, SIRT1-549-O with 500 mM free GlcNAc, anti-SIRT1 and anti-actin antibodies. (B) The lysates of NCI-H1299 cells transfected with empty or shOGT vectors were immunoblotted with SIRT1-549-O, SIRT1-549-O with free GlcNAc, anti-SIRT1, RL2, anti-OGT and anti-tubulin antibodies. (C) The lysates of NCI-H1299 cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O supplied with 1.5 nM O-GlcNAcylated SIRT1 peptide, anti-SIRT1 and anti-actin antibodies. (D) The lysates of SIRT1-WT and SIRT1-S549A cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O, anti-SIRT1, RL2 and anti-actin antibodies. Fig. 2. View largeDownload slide The SIRT1-549-O antibody is specific to the O-GlcNAcylated S549 site in human SIRT1. (A) The lysates of NCI-H1299 cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O, SIRT1-549-O with 500 mM free GlcNAc, anti-SIRT1 and anti-actin antibodies. (B) The lysates of NCI-H1299 cells transfected with empty or shOGT vectors were immunoblotted with SIRT1-549-O, SIRT1-549-O with free GlcNAc, anti-SIRT1, RL2, anti-OGT and anti-tubulin antibodies. (C) The lysates of NCI-H1299 cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O supplied with 1.5 nM O-GlcNAcylated SIRT1 peptide, anti-SIRT1 and anti-actin antibodies. (D) The lysates of SIRT1-WT and SIRT1-S549A cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O, anti-SIRT1, RL2 and anti-actin antibodies. SIRT1-549-O antibody can be applied to enrich SIRT1 O-GlcNAcylated at S549 by immunoprecipitation The enrichment of O-GlcNAcylated SIRT1 is important for its biochemical study, so we examined whether the SIRT1-549-O antibody could be used for immunoprecipitation. HEK 293 T cells that were pretreated with or without TMG were lysed for the immunoprecipitation assays using SIRT1-549-O antibody. The immunoprecipitates and total cell lysates were subjected to WB assays using anti-SIRT1 and SIRT1-549-O antibodies, respectively. The detection of the total cell lysates revealed that TMG treatment elevated the O-GlcNAcylation at SIRT1 S549 but did not affect the level of the SIRT1 protein. Regarding the immunoprecipitates, both O-GlcNAcylation at S549 of SIRT1 and SIRT1 proteins were increased in the TMG-treated sample (Figure 3). These results demonstrated that the SIRT1-549-O antibody can be used for immunoprecipitation assays. Fig. 3. View largeDownload slide The SIRT1-549-O antibody can be used for the immunoprecipitation of SIRT1 O-GlcNAcylated at S549. The lysates of wtSIRT1 and SIRT1S549A cells were immunoprecipitated with the SIRT1-549-O antibody. Then, the immunoprecipitates and total cell lysates were detected by WB using anti-SIRT1 and SIRT1-549-O antibodies. Fig. 3. View largeDownload slide The SIRT1-549-O antibody can be used for the immunoprecipitation of SIRT1 O-GlcNAcylated at S549. The lysates of wtSIRT1 and SIRT1S549A cells were immunoprecipitated with the SIRT1-549-O antibody. Then, the immunoprecipitates and total cell lysates were detected by WB using anti-SIRT1 and SIRT1-549-O antibodies. SIRT1-549-O antibody can be used to detect O-GlcNAcylated mouse SIRT1 To examine whether the SIRT1-549-O antibody can be used for the detection of SIRT1 O-GlcNAcylation in other species, we compared the S549 O-GlcNAcylation site of human SIRT1 with the same region of proteins from some other mammals. The results suggested that the S549 O-GlcNAcylation site of the human SIRT1 is highly conserved (Figure 4A). Thus, we detected protein samples from NIH/3T3 mouse embryonic fibroblast cells that were treated with or without TMG and mouse liver tissues using WB with the SIRT1-549-O antibody. An expected band at the 120 kDa position was detected in the samples from both the NIH/3T3 cells (Figure 4B) and the mouse liver tissues (Figure 4C). Additionally, TMG treatment increased the intensities of the bands that were blotted by the SIRT1-549-O antibody, which agrees with the elevation of the global cellular O-GlcNAcylation blotted by the RL2 antibody (Figure 4B). Collectively, these results indicated that the SIRT1-549-O antibody can be applied for the detection of mouse SIRT1 O-GlcNAcylation. Fig. 4. View largeDownload slide The SIRT1-549-O antibody is specific to O-GlcNAcylated mouse SIRT1. (A) Sequence alignment of the peptide containing the S549 site of human SIRT1 and the corresponding regions from other mammalian SIRT1 proteins. (B, C) The lysates from NIH/3T3 cells pretreated with or without 2 μM TMG (B) or mouse liver (C) were immunoblotted with SIRT1-549-O, RL2 and anti-SIRT1 antibodies. Fig. 4. View largeDownload slide The SIRT1-549-O antibody is specific to O-GlcNAcylated mouse SIRT1. (A) Sequence alignment of the peptide containing the S549 site of human SIRT1 and the corresponding regions from other mammalian SIRT1 proteins. (B, C) The lysates from NIH/3T3 cells pretreated with or without 2 μM TMG (B) or mouse liver (C) were immunoblotted with SIRT1-549-O, RL2 and anti-SIRT1 antibodies. S549 O-GlcNAcylation of SIRT1 is increased under oxidative stress Many previous reports have demonstrated that both O-GlcNAc and SIRT1 can serve as stress sensors (Houtkooper et al. 2012; Groves et al. 2013; Raynes et al. 2013). According to our recent finding, the O-GlcNAcylation of SIRT1 is involved in the cellular stress response (Han et al. 2017), but there is no direct evidence for the elevation of SIRT1 O-GlcNAcylation at the S549 residue during the stress response. Here, the SIRT1-549-O antibody was used to confirm the previous findings. The results revealed that H2O2 stimulation elevated the O-GlcNAcylation at S549 of endogenous SIRT1 in NCI-H1299 cells (Figure 5A). Furthermore, NCI-H1299 cells that were transfected with wtSIRT1 or SIRT1S549A were examined. We found that oxidative stress stimuli enhanced the O-GlcNAcylation at S549 of wtSIRT1 but not SIRT1S549A (Figure 5B). Similar results were also observed when mouse cells (Figure 5C) and mouse hepatic tissues (Figure 5D) were used. Taken together, these results suggest that SIRT1-549-O is a potent tool for the study of the roles and regulatory mechanisms of O-GlcNAcylation at the SIRT1 S549 residue in both cellular and mouse models. Fig. 5. View largeDownload slide The S549 O-GlcNAcylation of SIRT1 is increased under oxidative stress. (A–C) The lysates of HEK 293 T cells (A), wtSIRT1 cells (B), SIRT1S549A cells (B), and NIH/3T3 cells (C) that were treated with or without 0.2 mM H2O2 for 30 min or the indicated times were immunoblotted with SIRT1-549-O, anti-SIRT1 and anti-actin antibodies. (D) The mice were intraperitoneally injected with 200 μL of 100 mM H2O2 for 30 min, and the lysates of the mouse livers were immunoblotted with SIRT1-549-O, anti-SIRT1 and anti-actin antibodies. Fig. 5. View largeDownload slide The S549 O-GlcNAcylation of SIRT1 is increased under oxidative stress. (A–C) The lysates of HEK 293 T cells (A), wtSIRT1 cells (B), SIRT1S549A cells (B), and NIH/3T3 cells (C) that were treated with or without 0.2 mM H2O2 for 30 min or the indicated times were immunoblotted with SIRT1-549-O, anti-SIRT1 and anti-actin antibodies. (D) The mice were intraperitoneally injected with 200 μL of 100 mM H2O2 for 30 min, and the lysates of the mouse livers were immunoblotted with SIRT1-549-O, anti-SIRT1 and anti-actin antibodies. Discussion Recently, we found that SIRT1 is O-GlcNAcylated and that the main O-GlcNAcylation site is Ser 549 (Han et al. 2017). It is essential to develop a method for the specific detection of the O-GlcNAcylation at S549 of SIRT1 for the clarification of its roles and regulatory mechanisms. Here, we described the generation and characterization of a rabbit polyclonal antibody, named SIRT1-549-O, which was specific to SIRT1 that is O-GlcNAcylated at the Ser 549 residue. This antibody can be used for immunoprecipitation and WB assays for the detection of both human and mouse SIRT1. Additionally, we provide direct evidence for the elevation of SIRT1 with an O-GlcNAcylation at the S549 residue under oxidative stress using this antibody. Site-specific O-GlcNAc antibodies could significantly influence the study of O-GlcNAcylation, but there are still very few of them. In one of the successful examples, Meaghan Morris et al. developed a site-specific antibody for tau that is O-GlcNAcylated at S400, and this antibody was used to compare the S400 O-GlcNAcylation of tau in wild-type and human amyloid precursor protein transgenic mice (Cameron et al. 2013). Currently, almost all of the existing site-specific O-GlcNAc antibodies can only be applied for WB assays. However, the SIRT1-549-O antibody can be used for the immunoprecipitation of O-GlcNAcylated SIRT1 and thus provides the possibility for the enrichment of O-GlcNAcylated SIRT1 for biochemical study. Additionally, we also attempted to use the SIRT1-549-O antibody for immunofluorescence, but it did not work well (data not shown). Because of the highly conserved region around S549 of mammalian SIRT1, the SIRT1-549-O antibody can also be used to detect mouse SIRT1 O-GlcNAcylation. In summary, these data highlight the power of this antibody for the study of SIRT1 O-GlcNAcylation and its functional regulation. As one of the most conserved mammalian sirtuins, SIRT1 plays essential roles in many biological processes, such as stress management and cytoprotection (Meaghan et al. 2015). Recently, we found that SIRT1 is O-GlcNAcylated. The O-GlcNAcylation of SIRT1 at the S549 site directly enhances its deacetylase activity but does not obviously affect its subcellular localization or protein stability. Moreover, oxidative, genotoxic and metabolic stress stimuli can enhance the O-GlcNAcylation and activity of SIRT1, but we were previously unable to confirm this elevation of SIRT1 O-GlcNAcylation was that at S549. Here, we provide direct evidence using the SIRT1-549-O antibody. As suggested by previous results, SIRT1 O-GlcNAcylation may be an evolutionarily conserved process that drives cellular homeostasis by invoking various stress response pathways. Therefore, it is of great significance to monitor changes in SIRT1 O-GlcNAcylation at S549 in response to genetic or pharmacological manipulation. This study indicates that the SIRT1-549-O antibody is a potent tool that can readily detect changes in SIRT1 O-GlcNAcylation at this epitope in cellular and mouse models. This study generated and characterized an SIRT1-549-O antibody that can be used in WB and IP assays to detect the S549 O-GlcNAcylation of SIRT1. This antibody not only provides an effective approach for understanding the roles and regulatory mechanisms of SIRT1 O-GlcNAcylation but also makes it possible to discover drugs that could regulate SIRT1 activity by modulating O-GlcNAcylation. Materials and methods Generation of the SIRT1-549-O antibody The SIRT1-549-O antibody was generated at ImmunoWay, Inc. Two peptides, O-GlcNAc SIRT1 (CTSPPD-(O-GlcNAc)S-SVIVTLLD) and unglycosylated SIRT1 (CTSPPDSSVIVTLLD), were synthesized to 90% purity and conjugated to keyhole limpet hemocyanin (KLH). Additionally, two 3-month-old New Zealand White rabbits were immunized with KLH-conjugated O-GlcNAc SIRT1 peptides employing a protocol of five subcutaneous injections per rabbit. Furthermore, each rabbit received a 300-μg injection of the final immunogen in Freund’s complete adjuvant (CFA) on day 0 followed by three 200-μg booster injections in Freund’s incomplete adjuvant (IFA) on Days 14, 28 and 42. The production bleeds were performed on Days 49, 59, 62 and 69. The purified antibody was the designated SIRT1-549-O antibody. The antiserum titre was determined by indirect ELISA. Furthermore, the antiserum was purified with an O-GlcNAc peptide chromatographic column or an unglycosylated peptide chromatographic column when the titre reached 1:20,000. Antigen binding affinity ELISA ELISA plates were coated with 50 μL/well of 1 μg/mL KLH-conjugated O-GlcNAc SIRT1 peptide or unglycosylated SIRT1 peptide in bicarbonate coating buffer overnight at 4°C. Additionally, antigen-coated plates were blocked with 100 μL/well of 1% BSA in TBS for 1 h at 37°C followed by thorough washes in TBST. Initially, 1 μg/mL 50 μL of 3-fold serial dilutions of the SIRT1-549-O antibody prepared in TBS containing 1% BSA was added to the ELISA plate and incubated for 1 h at 37°C. After three washes in TBST, the HRP-conjugated goat anti-rabbit IgG was added and incubated for 1 h at 37°C. Moreover, color development was performed with a substrate solution containing 3′3′,5′,5′-tetramethylbenzidine (TMB) and halted by the addition of 1 M H2SO4. The absorbance at 450 nm was measured. Cell culture The cell lines HEK 293T, NCI-H1299 and NIH/3T3 were obtained from the Stem Cell Bank, Chinese Academy of Sciences. Specifically, the HEK 293T and NIH/3T3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (HyClone) with 10% fetal bovine serum (FBS; Invitrogen), and the NCI-H1299 cells were cultured in RPMI-1640 (HyClone) medium supplemented with 10% FBS. Plasmid The coding region of the wtSIRT1 with a 3xFlag-tag coding sequence at the 3′ terminal was cloned into MSCVpuro plasmids. Furthermore, the S549A SIRT1 mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene). The hygromycin B-resistant pLKO.1 vector was used to construct the SIRT1 shRNA vectors, and the targeting sequence was gctaagaatttcaggatta. Furthermore, the puromycin-resistant pLKO.1 vector was used to construct the OGT shRNA vectors, and the targeting sequence was cctaaattgatataagcatcc. Transient transfection and generation of stable cell lines According to the manufacturer’s instructions, the transient transfection of the NCI-H1299 cells was performed using Attractene Transfection reagents (QIAGEN), and the transient transfection of the HEK 293 T cells was performed using Lipofectamine 3000 (Invitrogen) lipofection. Furthermore, stable cell lines were obtained by retroviral or lentiviral infection, and selection was performed with puromycin (1 μg/mL) or hygromycin B (200 μg/mL) for 2 weeks. Western blotting The cells were lysed in dodecyl sulfate (SDS) lysis buffer (1% SDS, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM STZ, 40 mM GlcNAc, and Complete™ protease inhibitors [Roche]). Additionally, the lysate was resolved on 7.5% SDS polyacrylamide gels (SDS-PAGE), transferred to an Immobilon-FL PVDF membrane (Millipore) and immunoblotted with the indicated antibodies. The blots were probed with antibodies recognizing the following (the dilutions and clone/catalog numbers are in parentheses): SIRT1 (1:1000, CST), OGT (1:1000, Abcam), O-GlcNAc (RL2, 1:1000, Abcam), tubulin (1:2000, Abmart), and actin (1:5000, Sigma). Immunoprecipitation (IP) assay Cells were lysed in RIPA buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP40, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 10 μM PUGNAc [Sigma], 2 mM STZ [Sigma], 10 μM ThiametG [Sigma], 40 mM GlcNAc [Sigma], and Complete™ protease inhibitors [Roche]). Then, the cell lysates were gently mixed with specific antibodies and protein A beads for 3 h at 4°C. The immunoprecipitates were washed five times with lysis buffer, eluted with SDS sample buffer, and subjected to western blotting. Mice Female Balb/c mice (8–10 weeks old) were purchased from the Vital River Laboratory (Charles River) and then raised in a specific pathogen-free and air-conditioned animal facility. The mice were fed ad libitum with a standard diet (Shanghai Laboratory Animal Company) and kept under a 12-h light–dark cycle. The oxidative stress treatment was performed via an intraperitoneal injection of 200 μL of 100 mM H2O2, and three mice per group were used. The animal care and experimental procedures complied with the guidelines set up by the Ministry of Science and Technology of the People’s Republic of China. Statistical analysis The data were analyzed with Student’s t-tests using the SPSS 11.0 software program (SPSS). The data are presented as the means ± the standard errors of the mean (SEM). P < 0.05 was considered statistically significant. Funding This work was supported by the NSFC-Shandong Joint Fund (U1606403 and U1706210), the Scientific and Technological Innovation Project financially supported by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02), and also funded by Open Research Fund Program of Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology (Ocean University of China). Conflict of interest statement The authors declare that they have no conflicts of interest. Abbreviations HDAC histone deacetylases PTM post-translational modification TMB tetramethylbenzidine WB western blotting. References Bosch-Presegue L, Vaquero A. 2011. The dual role of sirtuins in cancer. Genes Cancer . 2: 648– 662. Google Scholar CrossRef Search ADS PubMed  Boutant M, Canto C. 2014. SIRT1 metabolic actions: Integrating recent advances from mouse models. Mol Metab . 3: 5– 18. 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Generation and characterization of a site-specific antibody for SIRT1 O-GlcNAcylated at serine 549

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Abstract

Abstract O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies thousands of proteins. However, the roles and mechanisms of O-GlcNAcylation have been clarified in only a few proteins, and one of the main reasons for this is the lack of site-specific anti-O-GlcNAc antibodies. Recently, we found that SIRT1, which is an NAD+-dependent deacetylase, is O-GlcNAcylated at the serine 549 site (S549) and plays a cytoprotective role under stress. However, the mechanism underlying the roles of SIRT1 O-GlcNAcylation remains unclear. Here, we describe a site-specific antibody for SIRT1 O-GlcNAcylated at S549, named SIRT1-549-O. This antibody can be used for immunoprecipitation and western blotting assays, and it can be used to recognize the endogenous levels of both human and mouse SIRT1 O-GlcNAcylation. Therefore, this antibody not only provides an effective method to further understand the roles of SIRT1 O-GlcNAcylation but also makes it possible to discover the genetic and pharmacological factors that could regulate SIRT1 activity by modulating its O-GlcNAcylation. O-GlcNAc, oxidative stress, SIRT1, site-specific antibody Introduction Post-translational modifications (PTMs) are covalent processing events that can change the properties of a protein by proteolytic cleavage or by the addition of a modifying group to one or more amino acids. These modifying groups consist of phosphate, glycans, ubiquitin, nitroso, methyl and acetyl, etc. PTMs not only increase the functional diversity of the proteome but also determine protein activity states, localizations, interactions with other proteins, and nearly all aspects of normal cell biology and pathogenesis. As is well known, phosphorylation refers to a ubiquitous protein post-translational modification. With the development and application of large numbers of site-specific antibodies for phosphorylation, the understanding of phosphorylation has been facilitated. Therefore, site-specific antibodies are powerful tools for the study of PTMs. Similar to phosphorylation, O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is also a dynamic and ubiquitous post-translational modification that occurs on the hydroxyl groups of the serine and/or threonine residues of nuclear and cytoplasmic proteins. The attachment of GlcNAc is catalyzed by O-GlcNAc transferase (OGT), which utilizes the uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) produced by the hexosamine biosynthesis pathway from glucose as the substrate (Kreppel et al. 1997). Reversely, O-GlcNAc moieties are removed from proteins by the glycoside hydrolase O-GlcNAcase (OGA) (Dong and Hart 1994). Currently, more than 4000 proteins with O-GlcNAcylation have been found. Indeed, O-GlcNAcylation participates in almost all biological processes and is involved in epigenetic regulation, protein translation, proteasomal degradation, signal transduction, stress responses, and cellular homeostasis (Hanover 2001). Although a few decades have passed since the discovery of O-GlcNAcylation, the study of O-GlcNAcylation is still progressing slowly. One of the main reasons for this is the lack of O-GlcNAcylation-specific antibodies. Some pan-O-GlcNAc antibodies have been developed including the antibodies CTD110.6 (Comer et al. 2001), RL2 (Snow et al. 1987), 18B10.C7(#3), 9D1.E4(#10), and 1F5.D6(#14) (Teo et al. 2010). Among these antibodies, CTD110.6 and RL2 are the most widely used. However, due to a certain degree of amino acid sequence dependency, these antibodies cannot detect all proteins with O-GlcNAcylation. Additionally, a few antibodies for site-specific protein O-GlcNAcylation have been reported, and all of these antibodies comprise anti-Tau (GlcNAc S400) (Cameron et al. 2013), anti-IRS2 (GlcNAc T1155), anti-H3 (GlcNAc T32) and anti-H4 (GlcNAc S47). These antibodies make it possible to study the functions and regulatory mechanisms of site-specific protein O-GlcNAcylation. SIRT1 is a NAD+-dependent deacetylase that belongs to the class III histone deacetylases (HDACs) (Smith et al. 2000). Moreover, SIRT1 plays a vital role in the regulation of metabolism (Boutant and Canto 2014), DNA repair (Choi and Mostoslavsky 2014), genomic stability (Oberdoerffer et al. 2008), the cell cycle (Brunet et al. 2004), cell survival and apoptosis (Luo et al. 2001), cellular senescence (Langley et al. 2002), and oncogenesis (Bosch-Presegue and Vaquero 2011) by deacetylating histones and various non-histone substrates. Recently, we found that SIRT1 is O-GlcNAcylated, which directly enhances the deacetylase activity of SIRT1, and the main O-GlcNAcylation site is serine 549 (S549) (Han et al. 2017). The O-GlcNAcylation of SIRT1 is elevated during genotoxic, oxidative and metabolic stress stimuli, and this process not only increases SIRT1 deacetylase activity but also protects cells from stress-induced apoptosis (Han et al. 2017). However, whether the roles of SIRT1 O-GlcNAcylation are due to the O-GlcNAcylation at S549 still needs to be confirmed. To achieve this, a site-specific antibody for SIRT1 O-GlcNAcylated at S549 will be helpful. Here, we described the generation and characterization of a rabbit polyclonal antibody, called SIRT1-549-O, which is specific for SIRT1 O-GlcNAcylated at S549. Furthermore, the utility of this antibody for biochemical applications is demonstrated. This antibody will be an effective tool for the study of the physiological and pathological effects of SIRT1 O-GlcNAcylation. Results SIRT1-549-O antibody is specific for SIRT1 peptide that is O-GlcNAcylated at Ser549 by ELISA To generate a rabbit polyclonal antibody against O-GlcNAcylated SIRT1 at S549, we synthesized two peptides spanning the sequence around S549, i.e., the O-GlcNAcylated SIRT1 peptide (CTSPPD-(O-GlcNAc)S-SVIVTLLD) and the unglycosylated SIRT1 peptide (CTSPPDSSVIVTLLD). The antibody antigen binding activity was tested by ELISA, and the results revealed that the SIRT1-549-O antibody exhibits highly specific binding activity with a calculated EC50 of 0.5 μg/mL towards the O-GlcNAcylated SIRT1 peptide but does not bind to the unglycosylated peptide (Figure 1). The lowest O-GlcNAc SIRT1 peptide-specific signal was detected at a 12 ng/mL antibody concentration. Altogether, these results indicated that the SIRT1-549-O antibody recognized the SIRT1 peptide in a manner that was dependent on the O-GlcNAcylation of SIRT1. Fig. 1. View largeDownload slide The SIRT1-549-O antibody is specific to the SIRT1 peptide O-GlcNAcylated at Ser549. ELISA with O-GlcNAcylated and unglycosylated SIRT1 peptides demonstrated the highly specific binding activity of SIRT1-549-O for the O-GlcNAcylated SIRT1 peptide. Fig. 1. View largeDownload slide The SIRT1-549-O antibody is specific to the SIRT1 peptide O-GlcNAcylated at Ser549. ELISA with O-GlcNAcylated and unglycosylated SIRT1 peptides demonstrated the highly specific binding activity of SIRT1-549-O for the O-GlcNAcylated SIRT1 peptide. SIRT1-549-O antibody is specific to O-GlcNAcylated human SIRT1 To examine whether the SIRT1-549-O antibody specifically recognizes the O-GlcNAcylated SIRT1 protein, the cell lysates of NCI-H1299 cells treated with/without the OGA inhibitor Thiamet G (TMG) were detected by western blotting (WB). Although several bands were blotted by the SIRT1-549-O antibody, there was a band with an apparent molecular weight (MW) of 120 kDa, which is the same molecular mass as the SIRT1 protein. Additionally, the intensity of the band was markedly elevated by the TMG treatment (Figure 2A, left panel). To verify whether the blotted bands were dependent on the O-GlcNAc moiety, 500 mM free GlcNAc was included while blotting with the SIRT1-549-O antibody. The results revealed that almost all of the bands were eliminated by free GlcNAc (Figure 2A, right panel). Additionally, OGT was silenced in the NCI-H1299 cells, and the cell lysates were blotted with the SIRT1-549-O antibody. As expected, OGT silencing effectively decreased the expression of OGT and the global cellular O-GlcNAcylation levels blotted by the RL2 antibody (Figure 2B). In line with the above results, OGT silencing significantly inhibited the intensity of the band blotted by the SIRT1-549-O antibody (Figure 2B). Collectively, these results demonstrated that the SIRT1-549-O antibody recognizes proteins depending on the O-GlcNAc moiety. To verify that the detected band at the position of 120 kDa was SIRT1 O-GlcNAcylated at S549, free O-GlcNAcylated SIRT1 peptide was added when the WB assays were performed using SIRT1-549-O antibody as detailed in figure 2A. The results revealed that the free peptide eliminated the band (Figure 2C). Furthermore, the NCI-H1299 cells exhibited silenced endogenous SIRT1 while expressing wild-type SIRT1 (wtSIRT1) or the mutant SIRT1 (SIRT1S549A, the Ser 549 was mutated to Ala); these findings were established and verified as previously reported (Han et al. 2017) and used to clarify the specificity of the SIRT1-549-O antibody. The results indicated that the mutation of S549 markedly decreased the intensity of the band (Figure 2D). The residual bands should indicate the detection of the endogenous SIRT1 that is not completely silenced. Fig. 2. View largeDownload slide The SIRT1-549-O antibody is specific to the O-GlcNAcylated S549 site in human SIRT1. (A) The lysates of NCI-H1299 cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O, SIRT1-549-O with 500 mM free GlcNAc, anti-SIRT1 and anti-actin antibodies. (B) The lysates of NCI-H1299 cells transfected with empty or shOGT vectors were immunoblotted with SIRT1-549-O, SIRT1-549-O with free GlcNAc, anti-SIRT1, RL2, anti-OGT and anti-tubulin antibodies. (C) The lysates of NCI-H1299 cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O supplied with 1.5 nM O-GlcNAcylated SIRT1 peptide, anti-SIRT1 and anti-actin antibodies. (D) The lysates of SIRT1-WT and SIRT1-S549A cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O, anti-SIRT1, RL2 and anti-actin antibodies. Fig. 2. View largeDownload slide The SIRT1-549-O antibody is specific to the O-GlcNAcylated S549 site in human SIRT1. (A) The lysates of NCI-H1299 cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O, SIRT1-549-O with 500 mM free GlcNAc, anti-SIRT1 and anti-actin antibodies. (B) The lysates of NCI-H1299 cells transfected with empty or shOGT vectors were immunoblotted with SIRT1-549-O, SIRT1-549-O with free GlcNAc, anti-SIRT1, RL2, anti-OGT and anti-tubulin antibodies. (C) The lysates of NCI-H1299 cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O supplied with 1.5 nM O-GlcNAcylated SIRT1 peptide, anti-SIRT1 and anti-actin antibodies. (D) The lysates of SIRT1-WT and SIRT1-S549A cells treated with or without 2 μM TMG were immunoblotted with SIRT1-549-O, anti-SIRT1, RL2 and anti-actin antibodies. SIRT1-549-O antibody can be applied to enrich SIRT1 O-GlcNAcylated at S549 by immunoprecipitation The enrichment of O-GlcNAcylated SIRT1 is important for its biochemical study, so we examined whether the SIRT1-549-O antibody could be used for immunoprecipitation. HEK 293 T cells that were pretreated with or without TMG were lysed for the immunoprecipitation assays using SIRT1-549-O antibody. The immunoprecipitates and total cell lysates were subjected to WB assays using anti-SIRT1 and SIRT1-549-O antibodies, respectively. The detection of the total cell lysates revealed that TMG treatment elevated the O-GlcNAcylation at SIRT1 S549 but did not affect the level of the SIRT1 protein. Regarding the immunoprecipitates, both O-GlcNAcylation at S549 of SIRT1 and SIRT1 proteins were increased in the TMG-treated sample (Figure 3). These results demonstrated that the SIRT1-549-O antibody can be used for immunoprecipitation assays. Fig. 3. View largeDownload slide The SIRT1-549-O antibody can be used for the immunoprecipitation of SIRT1 O-GlcNAcylated at S549. The lysates of wtSIRT1 and SIRT1S549A cells were immunoprecipitated with the SIRT1-549-O antibody. Then, the immunoprecipitates and total cell lysates were detected by WB using anti-SIRT1 and SIRT1-549-O antibodies. Fig. 3. View largeDownload slide The SIRT1-549-O antibody can be used for the immunoprecipitation of SIRT1 O-GlcNAcylated at S549. The lysates of wtSIRT1 and SIRT1S549A cells were immunoprecipitated with the SIRT1-549-O antibody. Then, the immunoprecipitates and total cell lysates were detected by WB using anti-SIRT1 and SIRT1-549-O antibodies. SIRT1-549-O antibody can be used to detect O-GlcNAcylated mouse SIRT1 To examine whether the SIRT1-549-O antibody can be used for the detection of SIRT1 O-GlcNAcylation in other species, we compared the S549 O-GlcNAcylation site of human SIRT1 with the same region of proteins from some other mammals. The results suggested that the S549 O-GlcNAcylation site of the human SIRT1 is highly conserved (Figure 4A). Thus, we detected protein samples from NIH/3T3 mouse embryonic fibroblast cells that were treated with or without TMG and mouse liver tissues using WB with the SIRT1-549-O antibody. An expected band at the 120 kDa position was detected in the samples from both the NIH/3T3 cells (Figure 4B) and the mouse liver tissues (Figure 4C). Additionally, TMG treatment increased the intensities of the bands that were blotted by the SIRT1-549-O antibody, which agrees with the elevation of the global cellular O-GlcNAcylation blotted by the RL2 antibody (Figure 4B). Collectively, these results indicated that the SIRT1-549-O antibody can be applied for the detection of mouse SIRT1 O-GlcNAcylation. Fig. 4. View largeDownload slide The SIRT1-549-O antibody is specific to O-GlcNAcylated mouse SIRT1. (A) Sequence alignment of the peptide containing the S549 site of human SIRT1 and the corresponding regions from other mammalian SIRT1 proteins. (B, C) The lysates from NIH/3T3 cells pretreated with or without 2 μM TMG (B) or mouse liver (C) were immunoblotted with SIRT1-549-O, RL2 and anti-SIRT1 antibodies. Fig. 4. View largeDownload slide The SIRT1-549-O antibody is specific to O-GlcNAcylated mouse SIRT1. (A) Sequence alignment of the peptide containing the S549 site of human SIRT1 and the corresponding regions from other mammalian SIRT1 proteins. (B, C) The lysates from NIH/3T3 cells pretreated with or without 2 μM TMG (B) or mouse liver (C) were immunoblotted with SIRT1-549-O, RL2 and anti-SIRT1 antibodies. S549 O-GlcNAcylation of SIRT1 is increased under oxidative stress Many previous reports have demonstrated that both O-GlcNAc and SIRT1 can serve as stress sensors (Houtkooper et al. 2012; Groves et al. 2013; Raynes et al. 2013). According to our recent finding, the O-GlcNAcylation of SIRT1 is involved in the cellular stress response (Han et al. 2017), but there is no direct evidence for the elevation of SIRT1 O-GlcNAcylation at the S549 residue during the stress response. Here, the SIRT1-549-O antibody was used to confirm the previous findings. The results revealed that H2O2 stimulation elevated the O-GlcNAcylation at S549 of endogenous SIRT1 in NCI-H1299 cells (Figure 5A). Furthermore, NCI-H1299 cells that were transfected with wtSIRT1 or SIRT1S549A were examined. We found that oxidative stress stimuli enhanced the O-GlcNAcylation at S549 of wtSIRT1 but not SIRT1S549A (Figure 5B). Similar results were also observed when mouse cells (Figure 5C) and mouse hepatic tissues (Figure 5D) were used. Taken together, these results suggest that SIRT1-549-O is a potent tool for the study of the roles and regulatory mechanisms of O-GlcNAcylation at the SIRT1 S549 residue in both cellular and mouse models. Fig. 5. View largeDownload slide The S549 O-GlcNAcylation of SIRT1 is increased under oxidative stress. (A–C) The lysates of HEK 293 T cells (A), wtSIRT1 cells (B), SIRT1S549A cells (B), and NIH/3T3 cells (C) that were treated with or without 0.2 mM H2O2 for 30 min or the indicated times were immunoblotted with SIRT1-549-O, anti-SIRT1 and anti-actin antibodies. (D) The mice were intraperitoneally injected with 200 μL of 100 mM H2O2 for 30 min, and the lysates of the mouse livers were immunoblotted with SIRT1-549-O, anti-SIRT1 and anti-actin antibodies. Fig. 5. View largeDownload slide The S549 O-GlcNAcylation of SIRT1 is increased under oxidative stress. (A–C) The lysates of HEK 293 T cells (A), wtSIRT1 cells (B), SIRT1S549A cells (B), and NIH/3T3 cells (C) that were treated with or without 0.2 mM H2O2 for 30 min or the indicated times were immunoblotted with SIRT1-549-O, anti-SIRT1 and anti-actin antibodies. (D) The mice were intraperitoneally injected with 200 μL of 100 mM H2O2 for 30 min, and the lysates of the mouse livers were immunoblotted with SIRT1-549-O, anti-SIRT1 and anti-actin antibodies. Discussion Recently, we found that SIRT1 is O-GlcNAcylated and that the main O-GlcNAcylation site is Ser 549 (Han et al. 2017). It is essential to develop a method for the specific detection of the O-GlcNAcylation at S549 of SIRT1 for the clarification of its roles and regulatory mechanisms. Here, we described the generation and characterization of a rabbit polyclonal antibody, named SIRT1-549-O, which was specific to SIRT1 that is O-GlcNAcylated at the Ser 549 residue. This antibody can be used for immunoprecipitation and WB assays for the detection of both human and mouse SIRT1. Additionally, we provide direct evidence for the elevation of SIRT1 with an O-GlcNAcylation at the S549 residue under oxidative stress using this antibody. Site-specific O-GlcNAc antibodies could significantly influence the study of O-GlcNAcylation, but there are still very few of them. In one of the successful examples, Meaghan Morris et al. developed a site-specific antibody for tau that is O-GlcNAcylated at S400, and this antibody was used to compare the S400 O-GlcNAcylation of tau in wild-type and human amyloid precursor protein transgenic mice (Cameron et al. 2013). Currently, almost all of the existing site-specific O-GlcNAc antibodies can only be applied for WB assays. However, the SIRT1-549-O antibody can be used for the immunoprecipitation of O-GlcNAcylated SIRT1 and thus provides the possibility for the enrichment of O-GlcNAcylated SIRT1 for biochemical study. Additionally, we also attempted to use the SIRT1-549-O antibody for immunofluorescence, but it did not work well (data not shown). Because of the highly conserved region around S549 of mammalian SIRT1, the SIRT1-549-O antibody can also be used to detect mouse SIRT1 O-GlcNAcylation. In summary, these data highlight the power of this antibody for the study of SIRT1 O-GlcNAcylation and its functional regulation. As one of the most conserved mammalian sirtuins, SIRT1 plays essential roles in many biological processes, such as stress management and cytoprotection (Meaghan et al. 2015). Recently, we found that SIRT1 is O-GlcNAcylated. The O-GlcNAcylation of SIRT1 at the S549 site directly enhances its deacetylase activity but does not obviously affect its subcellular localization or protein stability. Moreover, oxidative, genotoxic and metabolic stress stimuli can enhance the O-GlcNAcylation and activity of SIRT1, but we were previously unable to confirm this elevation of SIRT1 O-GlcNAcylation was that at S549. Here, we provide direct evidence using the SIRT1-549-O antibody. As suggested by previous results, SIRT1 O-GlcNAcylation may be an evolutionarily conserved process that drives cellular homeostasis by invoking various stress response pathways. Therefore, it is of great significance to monitor changes in SIRT1 O-GlcNAcylation at S549 in response to genetic or pharmacological manipulation. This study indicates that the SIRT1-549-O antibody is a potent tool that can readily detect changes in SIRT1 O-GlcNAcylation at this epitope in cellular and mouse models. This study generated and characterized an SIRT1-549-O antibody that can be used in WB and IP assays to detect the S549 O-GlcNAcylation of SIRT1. This antibody not only provides an effective approach for understanding the roles and regulatory mechanisms of SIRT1 O-GlcNAcylation but also makes it possible to discover drugs that could regulate SIRT1 activity by modulating O-GlcNAcylation. Materials and methods Generation of the SIRT1-549-O antibody The SIRT1-549-O antibody was generated at ImmunoWay, Inc. Two peptides, O-GlcNAc SIRT1 (CTSPPD-(O-GlcNAc)S-SVIVTLLD) and unglycosylated SIRT1 (CTSPPDSSVIVTLLD), were synthesized to 90% purity and conjugated to keyhole limpet hemocyanin (KLH). Additionally, two 3-month-old New Zealand White rabbits were immunized with KLH-conjugated O-GlcNAc SIRT1 peptides employing a protocol of five subcutaneous injections per rabbit. Furthermore, each rabbit received a 300-μg injection of the final immunogen in Freund’s complete adjuvant (CFA) on day 0 followed by three 200-μg booster injections in Freund’s incomplete adjuvant (IFA) on Days 14, 28 and 42. The production bleeds were performed on Days 49, 59, 62 and 69. The purified antibody was the designated SIRT1-549-O antibody. The antiserum titre was determined by indirect ELISA. Furthermore, the antiserum was purified with an O-GlcNAc peptide chromatographic column or an unglycosylated peptide chromatographic column when the titre reached 1:20,000. Antigen binding affinity ELISA ELISA plates were coated with 50 μL/well of 1 μg/mL KLH-conjugated O-GlcNAc SIRT1 peptide or unglycosylated SIRT1 peptide in bicarbonate coating buffer overnight at 4°C. Additionally, antigen-coated plates were blocked with 100 μL/well of 1% BSA in TBS for 1 h at 37°C followed by thorough washes in TBST. Initially, 1 μg/mL 50 μL of 3-fold serial dilutions of the SIRT1-549-O antibody prepared in TBS containing 1% BSA was added to the ELISA plate and incubated for 1 h at 37°C. After three washes in TBST, the HRP-conjugated goat anti-rabbit IgG was added and incubated for 1 h at 37°C. Moreover, color development was performed with a substrate solution containing 3′3′,5′,5′-tetramethylbenzidine (TMB) and halted by the addition of 1 M H2SO4. The absorbance at 450 nm was measured. Cell culture The cell lines HEK 293T, NCI-H1299 and NIH/3T3 were obtained from the Stem Cell Bank, Chinese Academy of Sciences. Specifically, the HEK 293T and NIH/3T3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (HyClone) with 10% fetal bovine serum (FBS; Invitrogen), and the NCI-H1299 cells were cultured in RPMI-1640 (HyClone) medium supplemented with 10% FBS. Plasmid The coding region of the wtSIRT1 with a 3xFlag-tag coding sequence at the 3′ terminal was cloned into MSCVpuro plasmids. Furthermore, the S549A SIRT1 mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene). The hygromycin B-resistant pLKO.1 vector was used to construct the SIRT1 shRNA vectors, and the targeting sequence was gctaagaatttcaggatta. Furthermore, the puromycin-resistant pLKO.1 vector was used to construct the OGT shRNA vectors, and the targeting sequence was cctaaattgatataagcatcc. Transient transfection and generation of stable cell lines According to the manufacturer’s instructions, the transient transfection of the NCI-H1299 cells was performed using Attractene Transfection reagents (QIAGEN), and the transient transfection of the HEK 293 T cells was performed using Lipofectamine 3000 (Invitrogen) lipofection. Furthermore, stable cell lines were obtained by retroviral or lentiviral infection, and selection was performed with puromycin (1 μg/mL) or hygromycin B (200 μg/mL) for 2 weeks. Western blotting The cells were lysed in dodecyl sulfate (SDS) lysis buffer (1% SDS, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM STZ, 40 mM GlcNAc, and Complete™ protease inhibitors [Roche]). Additionally, the lysate was resolved on 7.5% SDS polyacrylamide gels (SDS-PAGE), transferred to an Immobilon-FL PVDF membrane (Millipore) and immunoblotted with the indicated antibodies. The blots were probed with antibodies recognizing the following (the dilutions and clone/catalog numbers are in parentheses): SIRT1 (1:1000, CST), OGT (1:1000, Abcam), O-GlcNAc (RL2, 1:1000, Abcam), tubulin (1:2000, Abmart), and actin (1:5000, Sigma). Immunoprecipitation (IP) assay Cells were lysed in RIPA buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP40, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 10 μM PUGNAc [Sigma], 2 mM STZ [Sigma], 10 μM ThiametG [Sigma], 40 mM GlcNAc [Sigma], and Complete™ protease inhibitors [Roche]). Then, the cell lysates were gently mixed with specific antibodies and protein A beads for 3 h at 4°C. The immunoprecipitates were washed five times with lysis buffer, eluted with SDS sample buffer, and subjected to western blotting. Mice Female Balb/c mice (8–10 weeks old) were purchased from the Vital River Laboratory (Charles River) and then raised in a specific pathogen-free and air-conditioned animal facility. The mice were fed ad libitum with a standard diet (Shanghai Laboratory Animal Company) and kept under a 12-h light–dark cycle. The oxidative stress treatment was performed via an intraperitoneal injection of 200 μL of 100 mM H2O2, and three mice per group were used. The animal care and experimental procedures complied with the guidelines set up by the Ministry of Science and Technology of the People’s Republic of China. Statistical analysis The data were analyzed with Student’s t-tests using the SPSS 11.0 software program (SPSS). The data are presented as the means ± the standard errors of the mean (SEM). P < 0.05 was considered statistically significant. Funding This work was supported by the NSFC-Shandong Joint Fund (U1606403 and U1706210), the Scientific and Technological Innovation Project financially supported by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02), and also funded by Open Research Fund Program of Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology (Ocean University of China). Conflict of interest statement The authors declare that they have no conflicts of interest. Abbreviations HDAC histone deacetylases PTM post-translational modification TMB tetramethylbenzidine WB western blotting. References Bosch-Presegue L, Vaquero A. 2011. The dual role of sirtuins in cancer. Genes Cancer . 2: 648– 662. Google Scholar CrossRef Search ADS PubMed  Boutant M, Canto C. 2014. SIRT1 metabolic actions: Integrating recent advances from mouse models. Mol Metab . 3: 5– 18. 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GlycobiologyOxford University Press

Published: Apr 24, 2018

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