Pol ι

Cells were harvested and lysed with M-PER lysis buffer (Thermo Scientific) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Beyotime Biotechnology) for 30 min at 4°C. Protein concentration was determined using the BCA protein quantification kit (Beyotime Biotechnology). Equal amounts of the proteins were separated by SurePAGE™ precast gels with a linear gradient between 4%-20% (GenScript, Nanjing, China) and transferred to PVDF membranes (Millipore, Billerica, MA, USA) by eBlot^® L1 protein transfer system (GenScript). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against β-actin (Beyotime Biotechnology), Pol ι (Proteintech, Rosemont, IL, USA), G6PD (Abcam, Cambridge, MA, USA), OGT (Proteintech), O-GlcNAc (Invitrogen Life Technologies, Carlsbad, CA, USA), Erk and p-Erk (Cell Signaling Technology, Danvers, Massachusetts, USA) at 4°C overnight. After washing with TBST three times, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (MultiSciences, Hangzhou, China). High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China) was applied for band visualization. Images of the protein bands were collected by Tanon-5200 Chemiluminescent Imaging System (Tanon). β-actin expression was served as a loading control.

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.