Antirabbit or antimouse igg

The primary antibodies below were utilized: myosin heavy chain (MHC; R&D Systems, USA, MAB4470), slow MHC (Abcam, ab185967), fast MHC (Abcam, ab91506), MHC IIa (Abcam, ab124937), MHC IIb (Abcam, ab221149), myoglobin (Santa Cruz, USA, sc-393020), troponin I-ss (Santa Cruz, sc-514899), TOM20 (Abcam, UK, ab186735), TIM23 (Abcam, ab230253), SESN2 (Abcam, ab178518), AMPK (Cell Signaling Technology [CST], USA, 5831), phospho-AMPK (CST, 50081), PGC-1α (Abcam, ab106814), HIF-2α (Abcam, ab109616), GAPDH (Protein Technology, PRC, 60004-1-Ig). The primary antibody dilution factors were 1:3000 (R&D Systems), 1:5000 (Protein Technology), or 1:1000 (Abcam and CST).
In short, the protein was abstracted via RIPA lysis buffering solution to which 1% protease suppressor was added (Roche, USA, 11206893001). The same quantity of protein (10–50 μg) was isolated via 10% SDS–PAGE, and afterwards moved onto nitrocellulose films (Merck Millipore, USA, Z358657). The films were subjected to blockade with 5% w/v BSA prior to cultivation with the first antibodies under 4 °C overnight. Subsequently, the films were cultivated with second antibodies conjugated with antirabbit or antimouse IgG (Abcam) for 60 min under ambient temperature and visualized via the Immobilon ECL matrix tool (Merck Millipore, WBKLS0050).

Tissues and cells were placed in ice-cold lysis buffer (50 mM Tris, 5 mM EDTA, 250 mM NaCl, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mM sodium orthovanadate), homogenized, and incubated in ice for 20 min. Samples were centrifuged at 11,000 g for 10 min, supernatants were collected, and the protein concentration was measured with a BCA Protein Assay Kit (Pierce). Equal amounts of protein from colonic tissues or cell lines were loaded onto SDS-PAGE gels. After electrophoresis and transference, membranes were blocked with 5% nonfat dry milk in TBST and incubated overnight at 4°C with primary antibodies (LC3B 1 : 400, Abcam; ATG16L1 1 : 100, Abgent; p65 1 : 200, Santa-Cruze; β-actin 1 : 2000, Abcam; GAPDH 1 : 2000, Abcam). Subsequently, membranes were incubated with secondary antibody (anti-rabbit or anti-mouse IgG, 1 : 2000, Abcam). Signals were detected using WesternLumaxLight Sirius HRP substrate reagent (ZETA-Life, San Francisco, CA, USA). The bands were scanned using a ChemiDocXRS+Imaging System (Bio-Rad, Hercules, CA, USA) and quantified by Image Lab v5.2 software (Bio-Rad).

Protein extraction from the cell pellets was performed using a total protein extraction kit (Applygen Technologies, China) in accordance with the manufacturer's protocol. The protein concentrations were determined using a BCA protein assay reagent kit (Novagen, USA). Then, 20 μg of total protein samples were loaded and separated by SDS/PAGE electrophoresis and transferred to PVDF membranes (Millipore Corporation, USA). After blocking with 5% skim milk in TBST buffer (Tris-buffered saline, 0.1% Tween 20) at room temperature for 2 h, the membranes were incubated with the primary antibody (1:5000) at 4°C for 8 h. Subsequently, the membranes were incubated with a secondary antibody (1:5000, anti-rabbit, or anti-mouse IgG, Abcam, USA). Then, the protein level on the blot was detected using the Western Bright ECL kit (Bio-Rad Laboratories, USA). The following antibodies against the target proteins were used: rabbit monoclonal anti-calpain-1 antibody (ab108400, Abcam), rabbit monoclonal anti-caspase-3 antibody (9664, Cell Signaling Technology, USA), mouse monoclonal anti-caspase-8 antibody (9496, Cell Signaling Technology), and rabbit polyclonal anti-GAPDH antibody (ab9485, Abcam). Detection of GAPDH was used as the internal control.