Western Blot Analysis of Muscle Proteins

As detailed formerly (Lee et al., 2019 (link)), total proteins (40 μg) underwent 10 or 12% SDS-polyacrylamide gel (SDS-PAGE) and were electroblotted onto polyvinylidene fluoride (PVDF) membranes. Then, the target protein was blocked and probed at 4°C for 12 h with primary antibodies against PGC-1α, Atrogin-1, or MuRF1 (rabbit polyclonal, 1:1000; Abcam, Cambridge, MA, United States), Beclin1, p62 (SQSTM1/Sequestome 1), LC3 (microtubule-associated protein-1 light chain 3, LC3), Bcl-2, Bax, AMPK, Mfn2, Drp1, PINK1, p-AMPK^Thr172, Akt, p-Akt^Ser473, mTOR, p-mTOR^Ser2448, FoxO3a, p-FoxO3a^Ser253 (rabbit polyclonal, 1:1000; Cell Signaling Technology, Danvers, MA, United States), and GAPDH (rabbit polyclonal, 1:5000; Cell Signaling Technology, Danvers, MA, United States). After being washed three times for 15 min in 0.1% TBS-T buffer, the membrane was incubated with goat horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10000, Cell Signaling Technology, Danvers, MA, United States) at room temperature for 1 h, and the target protein was detected by enhanced chemiluminescence (ECL) reagent imaged by ultra-sensitive fluorescence/chemiluminescence imaging system ChemiScope6300 (CLiNX Science Instruments, Shanghai, China).