Phospho kap1

Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor cocktail (VICMED, VPI012), and the cell lysates were then centrifuged at 14,000 × g at 4 °C for 15 min. Protein concentrations were quantified using the Bicinchoninic Acid protein assay kit (BCA; Beyotime, P0010). Total proteins (50 μg) were separated by SDS-PAGE and electro-transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). Then the membranes were blocked with TBST buffer (NaCl 150 mmol/L, Tris 10 mmol/L, Tween-20 0.05% (v/v) pH = 7.6) containing 5% nonfat milk powder at room temperature for 2 h. The membranes were then probed overnight at 4 °C with primary antibodies, including phospho-KAP1(1:1000, Abcam, ab70369), KAP1(1:1000, Cell Signaling Technology, #4123), PCNA (1:2000, Servicebio, GB11010), Bcl-2 (1:1000, ABclonal, A0208), Bax(1:1000, ABclonal, A0207) followed by incubation with corresponding HRP-conjugated secondary antibodies (1:5000, Proteintech, SA00001-2) at room temperature for 2 h. Proteins on the membranes were visualized by adding the enhanced chemiluminescence reagent (Millipore, USA). Band intensities were analyzed using Image J 1.25 software (National Institutes of Health, Bethesda, USA) and normalized to loading controls.