Horseradish peroxidase hrp conjugated anti rabbit or anti mouse secondary antibody

Cells were harvested and lysed in RIPA lysis buffer (Beyotime Biotechnology, China) containing protease inhibitors for 20 min at 4°C. The proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, MA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies targeting β-actin (Beyotime Biotechnology, China), Pol ι (Proteintech, USA), MMP-2, MMP-9, JNK (Abcam, USA) and p-JNK (Cell Signaling Technology, USA). The membranes were then incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (Beyotime Biotechnology, China). The protein bands were visualized using enhanced chemiluminescence (ECL, Beyotime Biotechnology, China). Endogenous β-actin protein expression was detected as the internal control for each sample.

Cells were harvested and lysed in the radio immunoprecipitation assay (RIPA) lysis buffer that contained protease inhibitors for 20 min at 4°C. The proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, US). After blocking with 5% nonfat milk in TBS-Tween-20 for 1 h at room temperature, the membranes were incubated with primary antibodies targeting β-actin (Beyotime Biotechnology, Nantong, Jiangsu, China) and MAP4 (Proteintech, Rosemont, IL, US). The membranes were incubated with a horseradish peroxidase- (HRP-) conjugated anti-rabbit or anti-mouse secondary antibody (Beyotime Biotechnology, Nantong, Jiangsu, China) for 2 h after washing three times with TBST. The proteins were visualized using the enhanced chemiluminescence (ECL) Western blot analysis system (Bio-Rad). β-actin protein expression was detected as the internal control.