Ffp26

Proteins in H9c2 cells were extracted by lysis buffer (P0013J, Beyotime). After determining the concentration with a BCA detection kit (P0012, Beyotime), the proteins were loaded and electrophoresed on 10% SDS polyacrylamide gel (P0012AC, Beyotime), and then transferred onto PVDF membranes (FFP26, Beyotime,). After using 5% non-fat milk to block membranes for 60 min at 37°C, the following primary antibodies were incubated with membranes overnight at 4°C: Bcl-2 (ab59348, 1:1000, 26 kDa, Abcam, USA), Bax (ab32503, 1:1000, 21 kDa, Abcam), Cleaved caspase-3 (ab49822, 1:500, 17 kDa, Abcam), iNOS (ab3523, 1:200, 135 kDa, Abcam), Cox-2 (ab15191, 1:250, 69 kDa, Abcam), CDK4 (ab199728, 1:2000, 34 kDa, Abcam), Cyclin D1 (ab16663, 1:25, 36 kDa, Abcam), HSP70 (ab181606, 1:1000, 70 kDa, Abcam), and GAPDH (ab181602, 1:10000, 36 kDa, Abcam). Following extensive washing, protein bands were incubated with the secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab205718, 1:2000, 42 kDa, Abcam) at 37°C for 2 h. The detection of signal was performed according to a standard ECL method (27), and analysis software (Image J 1.5i, National Institutes of Health, USA) was used for images to measured protein expression. GAPDH was used as housekeeping gene.

The cellular proteins were extracted form skeletal muscle cells and lysed by RIPA lysate (R0010, Solarbio, Peking, China). Equal amounts of protein were electrophoresed on sodium lauryl sulfate polyacrylamide gel (SDS-PAGE) with a loading of 20 μL per well. The protein was then transferred to an activated PVDF membrane (FFP26, Beyotime, Shanghai, China) and blocked with 5% skim milk powder (P0216, Beyotime, Shanghai, China) for 2 h. Appropriately diluted primary antibodies against Beclin-1 (Abcam, Cat# ab223372), p62 (Abcam, Cat# ab101266), p-mTOR (Abcam, Cat# ab109268), mTOR (Abcam, Cat# ab2732), p-AKT^Ser473 (Abcam, Cat# ab18206), p-IRS1^Ser616 (CST, Cat# 2386S), IRS (CST, Cat# 2382S), AKT (CST, Cat# 2938S), LC3 (Affinity, Cat# AF5402) and β-actin (Abcam, Cat# ab8227) were added and incubated at 4 °C overnight. After washing the membrane, the corresponding secondary antibody (Cat# A0208 and A0216, Beyotime, Shanghai, China) was added for incubation. Chemiluminescence detection was then carried out using an enhanced chemiluminescence reagent (P0018AS, Beyotime, Shanghai, China). After development and fixing treatment, the film was photographed by a gel imaging analysis system (UniCel Dxl800, Beckman Coulter, Pasadena, CA, USA).

The collected samples were denatured by heating them for 5 min at 95°C with SDS-PAGE Sample Loading Buffer (P0015L, Beyotime, China). For SDS-PAGE, equal amounts of protein (20 g total protein) were loaded, and the gel was run at two stages: first 60 V for 20 min and then 80 V for 90 min using the Bio-Rad Mini-Protean (1,658,001). After activating the PVDF (FFP26, Beyotime, China) with methanol for a few minutes, the protein from the gel was transferred to the PVDF at 200 mA for 90 min using Bio-Rad Min Trans-Blot (1703930). A blocking buffer was used to block the membrane for 15 min at room temperature (P0231, Beyotime, China). According to the manufacturer’s recommended ratio, the first antibodies were diluted with dilution buffer (P0023A, Beyotime, China) and used for overnight membrane incubation at 4°C. The membrane was washed in TBST (P0023C, Beyotime, China) three times for 5 min each time and then incubated for 1 h at room temperature with the recommended dilution of conjugated secondary antibody in the dilution buffer (P0023D, Beyotime, China). Again, TBST was used to wash the membrane three times for 5 min each, and then HRP substrate (Millipore, WBKLS0500) was added to the membrane. Images were taken using the chemiluminescence Gel Doc (Bio-Rad Gel Doc/Chemi Doc Imaging System). The antibodies used are listed in Supplementary Table S3.