Anti blm

The cells cultured on cover slips were fixed with 2% paraformaldehyde and 2% sucrose in 1×PBS for 10 min and then permeabilized with 1% NP-40. After pre-incubation with 5% BSA/PBS, cells were incubated first with the primary antibody and then with the secondary antibody in 1% BSA/PBS for 1 h at room temperature. The slides were mounted with anti-fade mounting medium with DAPI (Solarbio, China). The primary antibodies used were anti-γH2AX (1:500, Abcam), anti-DNA G-quadruplex (G4) antibody, clone 1H6 (1:50, Merck, USA), anti-Blm (1:100, Invitrogen), anti-Wrn (1:100, Invitrogen), anti-Recql4 (1:100, Invitrogen), anti-Ddx11 (1:100, Invitrogen).

Cells were harvested and lysed in RIPA buffer containing protease inhibitor cocktail (Roche, Switzerland). The 20 μg of total protein were separated by SDS-PAGE and then transferred to PVDF membrane. After blocking in 10% non-fat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4° C. The membranes were then incubated with horseradish peroxidase-labeled secondary antibodies, and visualized with ECL. The primary antibodies used were anti-Blm (1:1000, Invitrogen, USA), anti-Wrn (1:1000, Invitrogen), anti-Recql4 (1:1000, Invitrogen), anti-Timeless (1:1000, Invitrogen), anti-Ddx11 (1:1000, Invitrogen), anti-Pot1 (1:1000, Invitrogen), anti-Fancd2 (1:1000, Novus Biologicals, USA), anti-Terf1 (1:1000, Invitrogen), anti-Gins2 (1:500, Santa Cruz, USA), anti-Top2a (1:1000, Novus Biologicals), anti-Mcm7 (1:1000, Santa Cruz), anti-Mcm2 (1:1000, Abcam, UK), anti-PCNA (1:1000, Abcam), anti-H2B (1:1000, Abcam), anti-tubulin (1:5000, Proteintech, USA), anti-actin (1:1000, Santa Cruz).