Enhanced chemiluminescence substrate reaction kit

RIPA lysis was used to extract the total protein from the cells. Protein concentrations were quantified using a BCA Protein Assay Reagent kit (Thermo Fisher Scientific, Waltham, MA, United States). Subsequently, the protein extracts were separated utilizing SDS-PAGE and then transferred onto the PVDF membrane (Millipore, Boston, MA, United States). Then, the membranes were incubated with the primary antibody (anti-ORC6 antibody, 1:1,000, ab153993; anti-Bax antibody, 1:1,000, ab182733; anti-PCNA antibody, 1:1,000, ab92552; anti-MMP-2 antibody, 1:1,000, ab92536; anti-MMP-9 antibody, 1:1,000, ab76003, Abcam, Shanghai, China) at 4°C for 8 h. After the membranes were washed by TBST, the membranes were incubated with horseradish peroxidase-coupled secondary antibody (goat anti-rabbit IgG H&L, 1: 2,000, Beyotime, Shanghai, China) for 1 h at room temperature. Next, the membranes were washed with TBST again. Ultimately, the protein bands were developed with an enhanced chemiluminescence substrate reaction kit (Thermo Fisher Scientific, Waltham, MA, United States). GAPDH was employed as the internal control.

Cells were collected and treated with RIPA lysate. Total protein was extracted. Protein concentration was explored by the BCA kit. Protein was separated by SDS-PAGE gel and transferred to the nitrocellulose membrane. The transfer membrane was blocked with a blocking solution containing 5% bovine serum albumin for 2 h, and the primary antibody was kept at 4°C overnight. Secondary antibody was kept at room temperature for 1 h. Finally, the enhanced chemiluminescence substrate reaction kit (Thermo Scientific product) was used to analyze the gray value of each protein band.