Apobec3b

Cells were lysed in RIPA lysis buffer containing 1% PMSF (Beyotime, Shanghai, China) on ice for 30 min. After centrifugation, the supernatant was collected, and its concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific, Wilmington, DE, USA). Next, 20 μg protein was separated on 12% SDS/PAGE gel (ACE Biotechnology, Nanjing, China) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Mllipore, Billerica, MA, USA). The membranes were blocked for 2 h at room temperature in media supplemented with 5% skim milk, followed by overnight incubation with primary antibodies of APOBEC3B (1/1000, Novus, Colorado, USA) and GAPDH (1/10,000, Abcam, Cambridge, UK) at 4℃. The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Zen-BioScience, Chengdu, China) for 1.5 h at room temperature and then washed thrice. Finally, the Enhanced Chemiluminescence (ECL) Western Blot Detection Kit (Milipore) was used for protein visualization. GAPDH was used as the protein loading control.