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.