Western Ligand Blot Analysis
Samples were prepared in nonreducing buffer, heated at 85°C for 3 min, and loaded onto a 14% Tris-glycine polyacrylamide gel. Electrophoresis and transfer were as described for Western immunoblot analysis. After transfer, detection was performed as described by Hossenlopp et al.. The nitrocellulose membrane was incubated overnight at 4°C with 400 000 cpm 125I-labeled activin A. After washing, the membrane was exposed at – 80°C to Kodak x-ray film and two intensifying screens for 4 days.
The bands on the x-ray films for the Western and Northern blots were scanned using a Color One Scanner (Apple, Cupertino, CA) and analyzed using NIH-Image software. cialis professional online
Data are expressed as mean ± SEM of at least three separate experiments. Data were analyzed by ANOVA followed by the Scheffe’s test. ED50 and IC50 values were determined using the curve-fitting PRISM program (GraphPad, San Diego, CA).