Western Blot Analysis of DNA Damage Response

The methodology was described previously^{29 (link),30 (link)}. The anti-HSC70 antibody was purchased from Santa Cruz (Santa Cruz, CA). Anti-cyclin A2, anti-phospho-ATR (Thr-1981), anti-phospho-Chk1 (Ser-345), anti-phospho-CDK1/2 (Tyr-15), and anti-γ-H2AX (Ser-139) antibodies were from Cell Signaling (Danvers, MA). Anti-phospho-RPA32 (S4/S8) and anti-BRCA2 antibodies were from Bethyl Laboratories (Montgomery, TX). Anti-phospho-histone H1 antibody was from Millipore-Sigma (Burlington, MA). To avoid the cross-reaction of secondary antibodies with previously probed primary antibodies, blots were stripped and re-probed with next primary antibodies raised in a different species in an alternate fashion. All images were acquired and processed using the G: BOX gel documentation system and the GeneSnap software (Syngene, Frederick, MD). Brightness and contrast of images were applied equally across the entire gel/blot image and to controls. Images of protein bands were cropped and only brightness-adjusted using the PowerPoint software (Microsoft, Redmond, WA). Quantification of the protein band intensity was performed using the ImageJ software (NIH, Bethesda, MD).