Western blot
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), anti-Recql4 (1:1000, Invitrogen),
anti-Pot1 (1:1000, Invitrogen), anti-Terf1 (1:1000, Invitrogen),
anti-Mcm7 (1:1000, Santa Cruz), anti-Parp1 (1:1000, Abcam), anti-p53
(1:5000, Proteintech), anti-Rb (1:1000, BD pharmingen), anti-p130
(1:1000, Abcam), anti-E2F1 (1:1000, Novus), anti-Cdc2 (1:1000, CST),
anti-Cdk2 (1:1000, CST), anti-actin (1:1000, Santa Cruz).