Rat anti rpa32

Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then blocked with PBS containing 0.25% Triton X-100 (Sigma) and 3% BSA (Sigma) for 1 h at room temperature. The cells were then incubated with primary antibodies at 4 °C overnight, washed with PBS, and incubated with appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature in the dark. Nuclei were stained with DAPI (Sigma). Primary and secondary antibodies were diluted in PBS containing 3% BSA. Images were captured at room temperature using an Olympus microscope (IX71) or a confocal microscope (TCS SP5; Leica) with a 63× oil objective. The p-ATM intensity was quantified by software LAS AF Lite (Leica). Antibodies used for immunofluorescence are as follows: rabbit anti-E-cadherin (Cell Signaling, 1:200), mouse anti-albumin (R&D, 1:200), mouse anti-p-ATM (Ser1981) (Thermo, 1:200), rabbit anti-HA-Tag (Cell Signaling, 1:200), rat anti-RPA32 (Cell Signaling, 1:200), rabbit anti-53BP1 (Novus, 1:200), goat anti-Nanog (R&D, 1:100), mouse anti-SSEA1 (R&D, 1:100), Cy3-conjugated donkey anti-mouse/rabbit/rat/goat IgG (Jackson Laboratories, 1:1 000), Cy5-conjugated goat anti-rabbit/mouse IgG (Jackson Laboratories, 1:1 000).

The following antibodies were used in this study: mouse anti-β-actin (Abcam, Cambridge, UK; cat# ab6276), mouse anti-γH2AX-S139 (Millipore, Burlington, MA, USA; cat# 05-636), mouse anti-c-Myc (Santa Cruz, Dallas, TX, USA; cat# sc-42), rabbit anti-cleaved PARP Asp214 (Cell Signaling, Danvers, MA, USA; cat# 9541), rabbit anti-MTH1 (Novus Biologicals, Centennial, CO, USA; cat# NB100-109), rabbit anti-p53 pS15 (Cell Signaling; cat# 9284), mouse anti-p53 (Santa Cruz; cat# sc-126), mouse anti-GAPDH (Abcam; cat# ab8245), rat anti-RPA32 (Cell Signaling; cat# 2208).
The secondary antibodies used were: goat anti-rat Alexa Fluor^® 568 (Life Technologies, Carlsbad, CA, USA; cat# A-11077), goat anti-rat Alexa Fluor^® 647 (Life Technologies; cat# A-21247), IRDye^® 800CW donkey anti-rabbit (LI-COR, Lincoln, NE, USA; cat# 926-32213), IRDye^® 680RD donkey anti-rabbit (LI-COR; cat# 926-68073), IRDye^® 800CW donkey anti-mouse (LI-COR; cat# 926-32212), IRDye^® 680RD donkey anti-mouse (LI-COR; cat# 926-68072).