Anti prpa32 s4 s8

PDCLs were cultured as standard and seeded in 96-well plates at a concentration of 10⁴ cells per well. At 24 hours after seeding, cells were either left untreated or exposed to 4 Gy of ionizing radiation and processed for analysis at 2, 4, and 20 hours after exposure. Cells were stained with primary antibodies at a dilution of 1:1000 with anti-pRPA32 (S4/S8; Bethyl Laboratories, Montgomery, TX) and anti-γH2AX (Ser139; Merck, Kenilworth, NJ). Secondary antibodies used were Alexa 488 anti-mouse IgG (green) and Cy3 anti-rabbit IgG (Sigma-Aldrich). 4′,6-Diamidino-2-phenylindole (Life Technologies, Rockville, MD) was used as a nuclear stain. Confocal imaging was performed by using the Opera Phenix high-content screening system (PerkinElmer, Waltham, MA) at ×63 magnification using a water objective, at wavelengths of 405 nm (4′,6-Diamidino-2-phenylindole), 488 nm (Alexa), and 561 nm (Cy3). A minimum of 320 cells (median, 980; range, 322–1886), in 2 separate experiments, were analyzed for each time point. Image analysis was performed by using the Columbus Image data storage and analysis system (PerkinElmer). Statistical analysis was performed with GraphPad Prism 6.

AZD1775 and olaparib were purchased from Selleck Chemicals (Houston, TX). Reagents were formulated and stored according to manufacturer’s protocols for in vitro and in vivo experiments. Following antibodies were used: primary antibodies against caspase 3 (#9664), Bax (#5023), MRE11 (#4847), NBS1 (#14956), ATM (#2873), RAD51 (#8875), 53BP1 (#4937), γH2AX (#9718), cyclinB1 (#12231), CDC2 (#28439), pCDC2 (Y15; #4539), pHH3 (#53348), ATR (#2790), pATR (S428; #2853),Chk1 (#2360), pChk1 (S317; #12302), pChk1(S345, #2348), histone H3 (#4499) and secondary HRP-conjugated goat anti-rabbit (#7074) and anti-mouse antibodies (#7076) from Cell Signal Technology (CST, Danvers, MA); anti-RPA32 (#ab76420) from Abcam (Cambridge, MA); anti-pRPA32 (S4/S8, #A300-245A) and anti-pRPA32 (S33, #A300-246A) from Bethyl Laboratories (Montgomery, TX); anti-PARP1(#Sc-8007) from Santa Cruz (Dallas, TX); anti-PAR (Ab-1, 10H; #AM80) from Merck Millipore, (Darmstadt, Germany); and anti-β-actin (#A5441) from Sigma-Aldrich (St. Louis, MO).