Western blot and immunoprecipitation assays
Tissues and cell proteins were extracted in ice-cold RIPA lysis buffer consisting of 150 mM NaCl, 50 mM Tris-HCl, 0.5% sodium deoxycholate, 200 mM NaF, 200 mM PMSF, 1.0% NP40, 1 mM EDTA, and protease and phosphatase inhibitor cocktail (Life Technology, NY). The protein concentration in the supernatants was determined with a BCA Kit (Pierce, Rockford, IL). Then, 20 µg of total protein was separated by electrophoresis in a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA) for western blot analysis. The signals were detected with the following antibodies by following standard procedures: STAT3 (#9145,CST, MA), phosphorylated STAT3 (#9139,CST, MA), HSP90 (ab13492, Abcam, MA), myostatin (ab71808 , Abcam,MA), CD63 (ab8219,Abcam, MA), atrogin-1 (sc166806,Santa Cruz, MA), MuRF-1 (sc398608,Santa Cruz, TX), MHC (#MAB4470,R&D Systems, MN), GAPDH (ab181602,Abcam, MA).
For the immunoprecipitation assays, cells were lysed in WB/IP buffer, and proteins were immunoprecipitated with the indicated antibodies. Precleared Protein A/G Plus-Agarose beads (Life Technology, New York, NY) were incubated with the immunocomplexes overnight and washed three times with lysis buffer. The immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting.