Atrogin 1

Gastrocnemius muscle samples were collected from each group and protein lysates were prepared to determine the levels of muscle atrogenes [atrogin 1], and muscle ring finger protein (MuRF1). Relative expression of these proteins was detected by immunoblot analysis using specific antibodies againts atrogin 1 (Abcam; 1:1000 dilution), MuRF1 (Santa Cruz Biotechnology; 1:1000 dilution), and GAPDH (Merck Millipore; 1:1000 dilution). Densitometric analyses of immunoblots were performed using ImageJ software^{50 (link)}.

Commercial antibodies used for immunoblotting in this study were S100A8 (Santa Cruz Biotechnology, CA, USA); S100A9 (Genetex, Irvine, CA, USA); Atrogin-1 (Abcam, Cambridge, UK); MuRF1 (Abcam, Cambridge, UK) and GAPDH (Santa Cruz Biotechnology, CA, USA). Protein extracts were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane. For detection of target proteins, the skimmed milk blocked membranes were incubated with specified primary antibodies and corresponding secondary antibodies conjugated with horseradish peroxidase. The bound antibodies were detected using enhanced chemiluminescence reagent (Merck Millipore, Burlington, MA, USA). Quantification was performed by densitometry using ImageQuant version 5.2 software (GE Healthcare, Chicago, IL, USA).

40 μg of protein from heart tissue or cell lysates or 10 μg of protein from isolated membrane lysates were loaded onto SDS-polyacrylamide gels. After electrophoresis, the separate proteins were transferred onto Bio-Rad polyvinylidene difluoride membranes. After blocking in 5% nonfat milk for 1 h, the membranes were incubated with antibodies against NOX4 (1:1000 dilution; Abcam), Rac1 (1:1000 dilution; Abcam), MuRF1 and atrogin-1 (1:1000 dilution; Abcam), Na⁺/K⁺-ATPase (1:1000 dilution; Abcam), p67^phox (1:1000 dilution; Abcam), phosphorylation of Ser-345 and Ser-370 on p47^phox and total p47^phox (1:1000 dilution; Thermo Fisher Scientific Inc.), phosphorylated p38 and total p38 (1:1000 dilution; Cell Signaling Technology), phosphorylated ERK1/2 and total ERK1/2 (1:1000 dilution; Cell Signaling Technology), and GAPDH (1:5000 dilution; Cell Signaling Technology), respectively, followed by secondary relevant antibodies conjugated with horseradish peroxidase. The signals were then developed using an enhanced version of the chemiluminescence reaction.
The protein ladders were purchased from FroggaBio Inc. (Concord, Canada) for cultured cardiomyocytes (245, 180, 135, 100, 75, 63, 48, 35, 25, 20, 17, and 11 kDa) and Thermo Fisher Scientific China Co. Ltd. for heart tissue samples (170, 130, 100, 70, 55, 40, 35, 25, 15, and 10 kDa).

Antibodies against Atrogin-1 (Cat# ab74023), MuRF-1 (Cat# ab172479), NRF-1 (Cat# ab175932), TFAM (Cat# ab131607), Cytochrome C (Cyt C) (Cat# ab13575), p38 MAPK (Cat# ab197348), p-p38 MAPK (Cat# ab195049), PGC-1α (Cat# ab54481), and GAPDH (Cat# ab9484) were purchased from Abcam (Cambridge, United States). Antibody against COXIV (Cat# bs1533) was purchased from Bioss (Beijing, China). p38 MAPK inhibitor SB203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) was purchased from Med Chem Express (NJ, United States). Megestrol acetate (Cat# B1377) was purchased from Ape × Bio (Houston, United States).

The assays were performed as previously described [6 (link)]. The primary antibodies used were as follows: GDF-15 (Abcam, ab128958), TSG101 (Proteintech, 28283-1-AP), Atrogin1 (Abcam, ab168372), CD9 (ABclonal, A19027), CD63 (ABclonal, A5271), CD81 (ABclonal, A5270), Bax (Proteintech, 50599-2-Ig), Bcl-2 (Proteintech, A11025), MHC (DSHB, MF20), Caspase3 (ABcolonal, A0214), Cleaved caspase3 (Cell Signaling Technology, 9661), Anti-mouse (Multi Sciences, GAM0072) and anti-rabbit (Multi Sciences, GAR0072). ECL Chemiluminescent Kit (Thermo Fisher, 03781) was used to visualize the antibody-antigen interaction and chemical luminescence of membranes was detected by Amersham Imager 600 (GE).

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).