Ab151501

Cells were harvested and lysed, and the supernatants were collected. Protein (25 μg) was loaded into each well of a 10% or 15% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel. Separated proteins were transferred onto a polyvinylidene fluoride membrane, followed by blocking for 1 h at room temperature in 5% nonfat milk. Using a primary antibody, the blots were then incubated overnight at 4°C. Thereafter, the HRP-labeled secondary antibody (diluted 1:1000) was incubated with the membrane at 37°C for 1 h according to the dosage. Finally, the membranes were washed with tris-buffered saline containing 0.5% (v/v) Tween 20 three times and incubated with electrochemiluminescence (Millipore) for image scanning. The density of each protein band was analyzed. The primary antibodies used in this study were as follows: TRIM58 (1:1000; Ab90362, Abcam, UK), RECQL4 (1:1000; Ab192375, Abcam, UK), AKT (1:1000; #4685, CST, USA), p-AKT (1:000; #4060, CST, USA), RECQL (1:500; Ab151501, Abcam, UK), Cyclin D1(1:10,000; Ab134175, Abcam, UK), P21 (1:1000; Ab109199, Abcam, UK), and GAPDH (1:2000; #5174, CST, USA).

The antibodies anti-RECQ1 (ab151501, 1:200 dilution), anti-53BP1 (ab175933, 1:200 dilution), anti-RPA (ab2175, 1: 1000 dilution), anti-PARP1 (ab191217, 1:1000 dilution) and anti-BrdU (ab6326, 1:200), anti-RPA2-pS4/S8 (ab87277, 1:200 dilution) were purchased from Abcam. Antibodies anti-γH2AX (2577, 1:800 dilution), α-Tubulin (2144, 1:1000 dilution) and PCNA antibody (2586, 1:200 dilution) were purchased from Cell Signaling. Mouse anti-BrdU (347580, 1:40) was purchased from BD Biosciences. AF647 (A-21247, 1:1000) and AF488 (A-11001, 1:1000) were purchased from Thermo Fisher Scientific. BMN673 (S7048, Talazoparib) was purchased from Selleck Chemicals.