Tbst solution

The cells were incubated with RIPA buffer containing protease inhibitors (protease inhibitors: RIPA = 1:100, Solarbio, Guangzhou, China) for 15 min on ice to fully lyse the cells 48 h after transfection. Then, the cells were centrifuged at 12,000× g for 10 min at 4 °C, and the supernatant was removed. Total protein was then quantified using the BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated in 12% SDS-PAGE and transferred to nitrocellulose membranes (Whatman, Maidstone, UK), blocked with 5% skim milk powder for 1 h, and then incubated with primary antibody solution overnight at 4 °C. Subsequently, PVDF membranes were washed three times for 5 min with TBST solution (Beyotime) and then incubated with secondary antibody solution for 60 min at room temperature. Western immunoblotting results were analyzed using the Odyssey Fc system (LI-COR, Lincoln, NE, USA). Antibody information is as follows: actived-caspase-3 p17 polyclonal antibody (BS7004; Bioworld, MN, USA; 1: 500) cleaved caspase-8 (Asp391) (18C8) rabbit mAb (9496; Cell Signaling Technology, Danvers, MA, USA; 1:1000), anti-caspase-9 antibody [E23] (ab32539; Abcam, Cambridge, UK; 1:1000), rabbit anti-GAPDH (AB-P-R 001; Hangzhou Goodhere Biotechnology Co., Ltd., Hangzhou, China; 1:1500).

rBriefly, cells were washed twice with precooled PBS, mixed with the RIPA buffer (Solarbio, Beijing, China) containing 1% PMSF (Solarbio, Beijing, China), and then incubated on ice for 30 min. The cell lysates were centrifuged at 12,000 g for 10 min. The supernatant was mixed with the 5× SDS-PAGE loading buffer and then incubated at 95°C for 5 min. The extracted proteins were separated via SDS-PAGE and transferred to a PVDF membrane (400 mA, 30 min). The membrane was blocked with 5% skimmed milk powder for 1 h, then incubated with a primary antibody solution overnight at 4°C. Afterward, the PVDF membrane was washed three times with TBST solution (Beyotime, Shanghai, China) for 5 min and then incubated with the secondary antibody solution at room temperature for 60 min. The protein immunoblot results were analyzed using the Odyssey Fc system (LI-COR, Lincoln, NE, USA). β-Tubulin was used as an internal reference. The relative protein levels normalized the β-tubulin protein content. The antibody information is as follows: rabbit anti-β-tubulin (1:1,000 dilution; Bioss, Beijing, China), rabbit anti-MyoD1 (1:1,000 dilution; Bioss, Beijing, China), MyHC antibody (1:1,000 dilution; DHSB, IA, USA), goat anti-rabbit IgG H&L antibody (1:3,000 dilution; Bioss, Beijing, China), and goat anti-mouse IgG H&L antibody (1:3,000 dilution; Bioss, Beijing, China).