Protein Immunoblot Analysis

Tissue (skeletal muscle or solid organs) was homogenized in seven volumes of homogenization buffer (20 mmol/L HEPES, 2 mmol/L EGTA, 50 mmol/L sodium fluoride, 100 mmol/L potassium chloride, 0.2 mmol/L EDTA, and 1% vol/vol protease inhibitor cocktail; pH 7.4). Lysates were subject to SDS-PAGE on triple-wide gels; 20–40 µg protein was loaded per lane. Following electrophoresis, proteins were transferred onto PVDF membranes, blocked, and incubated with primary and secondary antibodies, as described previously (19, 34). The following primary antibodies were used: total eIF4E-binding protein 1 (4EBP1), 1:1,000; phospho-4EBP1 Thr70, 1:1,000; total ribosomal protein S6 kinase 1 (S6K1), 1:1,000; phospho-S6K1 Thr389, 1:2,000; total Akt, 1:1,000; phospho-Akt Thr308, 1:1,000; total eukaryotic initiation factor 2α (eIF2α), 1:2,000; phospho-eIF2α Ser51, 1:2,000; total eukaryotic elongation factor 2 (eEF2), 1:2,000; phospho-eEF2 Thr56, 1:2,000; atrogin-1, 1:1,000; muscle RING-finger protein 1 (MuRF1), 1:1,000; microtubule-associated protein 1 A/1B light chain I/II (LC3-I/II), 1:2,000, and GAPDH, 1:5,000. The dilution of the corresponding secondary antibody was 1:10,000 for Akt, phospho-Akt, eIF2α, phospho-eIF2α, eEF2, phospho-eEF2, and GAPDH and 1:5,000 for other targets. Immunoreactivity was visualized with chemiluminescence. Total protein abundance was normalized for loading with GAPDH, and phosphoprotein abundance was normalized to the corresponding total protein abundance after stripping and reprobing the same membranes with nonphosphospecific antibodies.