Hrp conjugated anti mouse or anti rabbit secondary antibody

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, Shanghai, China) containing protease and phosphatase inhibitors (Beyotime Biotechnology) for 20 min at 4°C. Equal amounts of the proteins were separated by SurePAGEᵀᴹ precast polyacrylamide gels with a gradient between 4-20% (GenScript, Nanjing, China) and transferred to PVDF membranes (Millipore). After blocking with 5% nonfat milk (BioRad, Hercules, CA, USA), the membranes were incubated with primary antibodies against β-actin, AKT, phospho-AKT (S473), phospho-AKT (T308), GSK-3β, phospho-GSK-3β (S9) (Multi Science) and ILK (Abcam). The membranes were then incubated with an HRP-conjugated anti-mouse or anti-rabbit secondary antibody (Multi Sciences). The protein bands were visualized using High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China). Images were collected using the Tanon-5200 Chemiluminescent Imaging System (Tanon). β-actin protein expression was detected as loading control for each sample.