A0310

The cells or tissues were treated with RIPA lysate, properly mixed, centrifuged, and the supernatant was added to electrophoresis loading buffer. The separated proteins in the gel were transferred to PVDF membranes, coated with primary antibody overnight, incubated with secondary antibody the next day, and then exposed and developed using a gel imaging system. Primary antibodies used were anti-VDAC1 (A19707; Abclonal), anti-β-Catenin (A0316; Abclonal), phosphorylated β-catenin (AP1076; Abclonal), anti-Cyclin D1 (A0310; Abclonal), anti-GAPDH (A19056; Abclonal).

Total protein was extracted from tissues and cells using RIPA lysis buffer (R0010; Solarbio) and protein concentration was quantified using BCA protein assay kit (PC0020; Solarbio). Twenty micrograms of total protein from each sample was subjected to SDS-PAGE using 10% polyacrylamide gels and transferred to PVDF membranes (IPVH00010; Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk and then incubated with primary antibodies against CYLD (1:1,000; 11110-1-AP, Proteintech), QKI (1:1,000; 13169-1-AP, Proteintech), poly (ADP-ribose) polymerase 1 (PARP-1) (1:1,000; 22999-1-AP, Proteintech), p27 (1:2,000; 25614-1-AP, Proteintech), E2F1 (1:1,000; 12171-1-AP, Proteintech), macroH2A1.1 (1:1,000; A7045, ABclonal, Wuhan, China), cyclin D (1:1,000; A0310, ABclonal), c-Fos (1:1,000; A0236, ABclonal), macroH2A1.2 (1:1,000; #4827, CST, Danvers, MA, USA), and GAPDH (1:10,000; 60004-1, Proteintech) at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated sheep anti-rabbit IgG or anti-mouse IgG antibody for 1 h at 37°C. The enhanced chemiluminescence kit (ECL, PE0010; Solarbio) was used to detect the signals of protein bands and normalized to GAPDH.