Anti rad51 pc 130

Cells were planted and grown on chamber slides. After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Then, slides were blocked with 10% goat serum and incubated with primary antibodies including anti-Rad51 (PC-130, EMD Millipore, 1:500) and anti-BRCA1 (sc-6954, Santa Cruz Biotechnology, 1:200) at 4°C overnight. Then, the slides were extensively washed with PBS and incubated with Alexa Fluor secondary antibodies (1:5000, Cell Signaling Technology) in the dark for 1 h. After washing with PBS and mounting with Prolong Gold antifade DAPI, the slides were assessed using a confocal microscope.

Briefly, U2 OS cells were collected and lysed using cell lysis buffer for WB and IP (P0013, Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride (PMSF, ST506, Beyotime, Shanghai, China) and protease inhibitor cocktail (Roche, Mannheim, Germany). Proteins were then electrophoresed using SDS-PAGE gels (Beyotime, Shanghai, China) and transferred to polyvinylidene difluoride membranes (PVDF, 0.22 μm, PALL-BSP0161, MD, USA). After blocking, membranes were incubated with primary antibodies and then with HRP-conjugated secondary antibodies at RT. The primary antibodies used were as follows: anti-β-actin (8H10D10, 1:1000; CST, MA, USA), anti-PRMT5 (07-405, 1:1000; Millipore, Darmstadt, Germany), anti-p21 (12D1, 1:1000; CST, MA, USA), anti-PCNA (D3H8P, 1:1000; CST, MA, USA), anti-p16 (92803S, 1:1000; CST, MA, USA), anti-γ-H2A.X (20E3, 1:1000; CST, MA, USA), anti-p53 (9282, 1:1000; CST, MA, USA), anti-TXNIP (D5F3E, 1:1000; CST, MA, USA), anti-TRIM21 (D1O1D, 1:1000; CST, MA, USA), anti-Lamin B1 (D9V6H, 1:1000; CST, MA, USA), and anti-RAD51 (PC130, 1:1000; Merck, Darmstadt, Germany). Secondary HRP-conjugated antibodies (1:1000) were purchased from CST (MA, USA). Immunoreactive bands were visualized using Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA).