First, 1 × 107 cells were washed with ice-cold PBS and lysed in 500 μL CHAPS buffer supplemented with protease inhibitor cocktail for 30 minutes at 4°C. Lysates were centrifuged at 20,000g for 10 minutes at 4°C. Next, 50 μL supernatants were aliquoted as input sample and supplemented with SDS sample buffer, then incubated for 10 minutes at 95°C. Protein G Dynabeads (Thermo Fisher Scientific, 10004D) were washed once with washing buffer to reduce nonspecific binding. Next, 450 μL supernatants were transferred and incubated with 20 μL Protein G Agarose Beads and rabbit polyclonal anti-RPA1 IgG (Abcam, ab241950) at 5 μg/mg of lysate overnight at 4°C. The beads were washed 3 times with washing buffer and incubated with 50 μL SDS sample buffer for 10 minutes at 95°C. The IP complex and 10 μg input samples were then subjected to Western blot analysis.

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