Total cellular RNA was isolated using the RNeasy Total RNA kit (Quiagen, Chatsworth, CA), and the aliquot of 3 fig was electrophoresed on a 0.66 M formaldehyde-agarose gel. The RNA samples were then transferred onto nylon membrane filters and hybridized at 68°C for 24 h with a 32P-labeled cRNA probe in a solution of 50% formamide, 6-strength SSPE (60 mM NaPQ4 [pH 7.0], 1.08 M NaCl, 6 mM EDTA), 0.5% SDS, 5-strength Denhardt’s solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA), 0.2 mg/ml yeast tRNA, and 0.2 mg/ml denatured salmon sperm DNA. The inh-a cRNA probe encoding a 166-basepair (bp) Hincll/Pst I segment of a rat inh-a clone was synthesized by in vitro transcription using [a-32P]UTP (650 Ci/mmol). The plasmid of pTRI-cyclophilin-rat (Ambion, Austin, TX) that contains 103 bp of the rat cyclophilin gene spanning exon 1 and 2 was used to make a rat cyclophilin cRNA probe. After the hybridization, the filters were washed with vigorous agitation in 300 mM NaCl, 30 mM sodium citrate, 0.1% SDS for 15 min at room temperature and then incubated in 15 mM NaCl, 1.5 mM sodium citrate, 0.1% SDS for 1 h at 65°C. Autoradiography was performed on a Kodak x-ray film (Eastman Kodak, Rochester, NY) with an intensifying screen at — 80°C. The same filter was used for rat Inh-a and cyclophilin Northern analysis. buy diabetes drugs
lodination of Activin A
Iodination was according to the chloramine T method. Activin A (3 jig in 45 (xl of 0.1 M phosphate buffer, pH 7.4) was incubated with 5 |a1 (500 (xCi) of 125I-labeled sodium (Amersham) for 1 min at room temperature. 125I-labeled activin A was separated from unlabeled radioactivity using a 10-ml Sephadex G-25 column (Pharmacia Biotech, Oiscataway, NJ) eluted with 0.05 M phosphate, 1% BSA-buffered saline, pH 7.4.