Rpa32

Whole cell lysates were prepared using RIPA cell lysis buffer (9803, Cell Signaling) and 1 mM PMSF (8553s, Cell Signaling) and western blots were performed using the following antibodies MYC (Cell Signaling), pKAP1 (S824), KAP1, pCHK1 (S345), CHK1, pRPA32 (S33), RPA32, γH2AX and Vinculin (sc-25336, Santa Cruz).

Cells were harvested in PBS and lysed for 30 min on ice in IP lysis buffer (200 mM NaCl, 0.75% Chaps, 50 mM Tris pH 8.0) or RIPA buffer (10 mM Tris-Cl (pH 8.0) 1 mM EDTA. 1% Triton X-100. 0.1% sodium deoxycholate. 0.1% SDS. 140 mM NaCl) (Hall et al., 2014 (link)), supplemented with protease inhibitors. Lysates were clarified by centrifugation (13,000 rpm for 10 min at 4°C) and the supernatants were collected. Protein samples were quantified with the Bradford assay and resolved by SDS-PAGE, transferred onto PVDF, and probed using the indicated primary antibodies. The membrane was stained with Alexa Fluor 680 or 800 anti-mouse/rabbit secondary antibody (Life Technologies) and visualized with the Licor Odyssey system. The following antibodies were used for staining: GAPDH (Santa Cruz, sc-47724); Flag (Santa Cruz, sc-51590); GFP (Abcam, Ab6556); HA (Sigma, H9658), RPA70 (Cell Signaling, 2267), RPA32 (Santa Cruz, sc-14692), SAMHD1 (Origene, TA502024), CtIP (14-1, a generous gift from Richard Baer) (Yu and Baer, 2000 (link)), BRCA2, RAD51 (Calbiochem, PC130), γH2AX (Cell Signaling, 2577 or Millipore, 05-636), 53BP1 (Bethyl, A300-273A), BrdU (BD Biosciences, 347580), MRE11 (Abcam, ab30725).