Phospho eif4ebp1

Total cell lysates were solubilized in ice-cold RIPA lysis buffer (Beyotime Biotechnology, P0013B, China) consisting of protease and phosphatase inhibitor cocktails (HY-K0010, HY-K0021, HY-K0022, MCE, United States). The membranes were blocked with 5% bovine albumin for 2 h at room temperature prior to incubation with the indicated primary antibodies. The signals were detected with the following antibodies following standard procedures: phosphorylated STAT3 (CST, #9139), Atrogin-1 (Satan Cruz, sc166806), MuRF-1 (Santa Cruz, sc398608), MHC (R&D Systems, #MAB4470), p62 (CST, #5114s), LC3 (Proteintech, 14600-1-AP), phospho-P70 S6K (Beyotime, AF5899), phospho-EIF4EBP1 (Beyotime, AF5806), phospho-mTOR (Beyotime, AF5869), GDF8/Myostatin (Proteintech, 19142-1-AP), iNOS (Affinity, AF0199), phospho-Akt (Ser473) (CST, #4060), phospho-AMPK (Abcam, ab133448), ATGL (CST, #2439), HSL (CST, #18381), UCP-1 (Satan Cruz, sc518171) and GAPDH (Proteintech, 60004-1-Ig). Subsequently, the membranes were washed and incubated for 2 h at room temperature with peroxidase-conjugated secondary antibodies (Bioworld). Following several washes, chemiluminescent images of immunostained bands on the membranes were recorded on X-ray films using the enhanced chemiluminescence (ECL, FUDE, FD8020, China) system according to the manufacturer’s instructions.

Total cell lysates were solubilized in ice-cold RIPA lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitor cocktails (MedChemExpress (Monmouth Junction, NJ, USA). Incubation with the primary antibodies was performed after blocking the membranes with 5% bovine albumin at room temperature for 2 h. Standard procedures were followed for the detection of the signals using the following antibodies: STAT3 (CST, #9145), phosphorylated STAT3 (CST, #9139), atrogin-1 (Satan Cruz, sc166806), MuRF-1 (Santa Cruz, sc398608), MHC (R&D Systems, #MAB4470), p62 (CST, #5114s), LC3 (Proteintech, 14600-1-AP), cleaved caspase3 (CST, #9664T), cleaved PARP (CST, #5625), phospho-P70 S6K (Beyotime, AF5899), phospho-EIF4EBP1 (Beyotime, AF5806), GLUT1/SLC2A1 rabbit mAb (Abclonal, A11727), Bdh1 rabbit pAb (Abclonal, A3763), HMGCS2 antibody (Affinity, DF14319), and GAPDH (Proteintech, 60004-1-Ig). Subsequently, at room temperature, the membranes were washed and incubated for 2 h with peroxidase-conjugated secondary antibodies (Bioworld). The enhanced chemiluminescence (ECL) system was used to record chemiluminescent images of immunostained bands on membranes on X-ray films, following the manufacturer’s protocol.