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METHODS Cell culture. NHTBE cells from passage 2 were grown on collagen gel-coated Transwell clear membranes (Costar, Cambridge, MA) in serum-free media containing various supplements, including 0.5 rig/ml epidermal growth factor, as previously described (6). The retinoid used was 5 X 10es M all trans-retinoic acid (Sigma, St. Louis, MO), unless otherwise indicated. In some experiments, retinol (Sigma) was used at 5 x lO-7 M RNA pzkfication. Total RNA was isolated from day 14 NHTBE cell cultures using Tri-Reagent (Molecular Research Center, Cincinnati, OH). Quality the RNA was assessed by electrophoresis 1 yg RNA on a 1.5% agarose gel containing 6.6% formaldehyde. Ethidium bromide was added to the RNA sample before loading on the gel to allow visualization the RNA. Total RNA from human tissues was obtained from Clontech (Palo Alto, CA). RTPCR. Oligonucleotide primers for RT-PCR were designed according to the published human MUC2 (Ref. 8; Genbank accession no. L21998, 5â-primer: TGCCTGGCCCTGTCTTTG; 3â-primer: CAGCTCCAGCATGAGTGC) and human MUC5AC (Ref. 15; Genbank accession no. UO6711, 5â-primer: TCCGGCCTCATCTTCTCC; 3â-primer: ACTTGGGCACTGGTGCTG) sequences and were obtained from Genosys (Woodlands, TX). Oligonucleotide amplimers for Pz-microglobulin (P2M) were purchased from Clontech Laboratories. RT-PCR reactions were performed on a PerkinL1023 S ARE LARGE-MOLECULAR WEIGHT, highly
AJP - Lung Cellular and Molecular Physiology – The American Physiological Society
Published: Dec 1, 1996
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