Anti iκbα p iκbα

After lysing cells with RIPA (Beyotime, Shanghai, China) to obtain protein samples, protein concentration was measured using the BCA method. After electrophoresis, 5% skimmed milk was blocked for 2 h at room temperature, the primary antibody was incubated overnight at 4 °C, and the secondary antibody was incubated for 1 h at room temperature; anti-p38/p-p38, anti-ERK/p-ERK, anti-JNK/p-JNK, anti-p65/p-p65, anti-IKKα/p-IKKα, anti-IκBα/p-IκBα, anti-Sirt1, anti-β-Actin, anti-β-Catenin were obtained from Cell Signalling Technology (1:1000 dilution); anti-PGC1α, anti-COX15, anti-NDUFV2, anti-ATP5D, anti-ATP5H were obtained from Proteintech Biotechnology (1:1000 dilution); anti-TFAM was obtained from Absin Bioscience (1:1000 dilution); anti-p-GSK3β/GSK3β was purchased from Santa Cruz Biotechnology (1:1000 dilution); Peroxidase-conjugated secondary antibody (1:4000 dilution) was used, and ECL reagent (Beyotime, Shinghai, China) was added at a 1:1 ratio to develop the image on a digital imaging system (Bio-Rad, USA).

Total protein was extracted using RIPA buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail. Protein concentrations were determined by the BCA protein assay. Total proteins (40 μg) were separated by SDS-PAGE and blotted onto PVDF membranes (Millipore, Massachusetts, USA). After blocking with 5% BSA in TBST, the membranes were incubated with the following primary antibodies at 4 °C overnight; anti-CD9, anti-CD63, anti-TSG101, anti-VDAC, anti-LAMP1 were obtained from Abcam (Abcam, USA); and anti-p38/p-p38, anti-ERK/p-ERK, anti-JNK/p-JNK, anti-p65/p-p65, anti-IKKα/p-IKKα, anti-Iκbα/p-Iκbα, anti-Sirt1, anti-GAPDH, anti-β-actin were obtained from Cell Signaling Technology (CST, USA); and anti-PGC1α, anti-cox15, anti-NDUFV2, anti-ATP5D, anti-ATP5H were obtained from Proteintech Biotechnology (Proteintech, USA); and anti-TFAM was obtained from Absin Bioscience (Absin, Shanghai, China); and anti-TOM20, anti-CALR were obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Thereafter, the membranes were incubated with HRP-conjugated anti-mouse and anti-rabbit IgG (1:4000, CST, USA), respectively, at room temperature for 1 h. ECL reagent was used to develop the membrane and signal was detected on a digital image system (Bio-Rad, USA).