DNA Damage Response Signaling Assays

Western blot and immunofluorescence were performed as described previously (9 (link), 32 (link)). The primary antibodies were anti-ATR (2790S, 1:800), anti-ATR (13934, 1:1000), anti-CHK1 (12908, 1:500), anti-phospho-CHK1 S317 (2344, 1:1000 for western blot; 1:150 for immunofluorescence), anti-WEE1 (sc-5285, 1:500), anti- pCDK1 (Y-15) (4539, 1:1000), anti-phospho-RPA2 Ser4/Ser8 (A300-245A, 1:2000 for western blot; 1:500 for immunofluorescence), anti-Rad51 (PC130, 1:1500), anti-Rad51 (sc-398587, 1:100), anti-γH2AX Ser139 (05-636, 1:1500), anti-53BP1 (NB100-304, 1:2500), anti-MUS81 (11018-1-AP, 1:1000), anti-Phospho-Histone H3 (Ser10) (66863-1-Ig, 1:2000), anti-phospho-RPA2 S33 (TA312067S, 1:1000), anti-CDK1 (CY5176, 1:1000) and anti-GAPDH (TA-08, 1:2000). Secondary antibodies included the goat anti-rabbit IgG horseradish peroxidase conjugated and goat anti-mouse IgG-horseradish peroxidase conjugated IgG (Pierce) in addition to the AlexaFluor 594-labeled goat anti-mouse IgG and AlexaFluor 488-labeled chicken anti-rabbit IgG (Molecular Probe). The images from the immunofluorescence assays were viewed at 100× magnification with a Zeiss Axio observer inverted fluorescence microscope (3858000984).