Phospho chk1 ser296

Mouse brain tissue was fixed in 4% paraformaldehyde in PBS overnight at 4°C and embedded in paraffin. Tissue sections (5 μm) underwent antigen retrieval in a citrate buffer before immunostaining with the following antibodies: Ki-67 (Leica, #NCL-Ki67p, 1:5000), cleaved caspase-3 (BD, #559565, 1:500), phospho-CHK1 Ser³⁴⁵ (Cell Signaling, #2348, 1:200), phospho-CHK1 Ser²⁹⁶ (Cell Signaling, #2349, 1:200), phospho-CDC2 Tyr¹⁵ (Cell Signaling, #4539, 1:200), γH2AX (Cell Signaling, #9718S, 1:500). Antibodies were detected using Elite ABC kit and NovaRED substrate, then counterstained with Gill’s hematoxylin (all Vector Laboratories). Positively-stained cells were quantified using a Nuance spectral unmixing camera and InForm software (Perkin Elmer), or Image J software (64 (link)) was used to apply a threshold limit to a minimum of three images per tumor and the average number of pixels above the threshold was determined from at least three independent animals per group.

Cells were cultured in the presence or absence of drugs as described. DMSO (0.1%) remained consistent across all samples. Cells were lysed 24 h after addition of the DNA-damaging agent in radioimmunoprecipitation buffer (150 mM sodium chloride, 50 mM Tris.HCl pH 8, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors (Roche). Protein (20 μg) was separated using 4–12% gradient Bis-Tris gels (Nupage) and transferred to nitrocellulose (BioRad). Antibodies were purchased from Cell Signaling and used at 1:1000: phospho-CHK1 Ser³⁴⁵ (#2348), phospho-CHK1 Ser²⁹⁶ (#2349), phospho-CHK2 Ser⁵¹⁶ (#2669), phospho-CHK2 Thr⁶⁸ (#2661), phospho-CDC2 Tyr¹⁵ (#4539), γH2AX (#9718), CHK1 (#2360), CHK2 (#6334), CDC2/CDK1 (#9116), and p53 (#2527). β-actin (#A1978, 1:5000, Sigma Aldrich) was used as a loading control. Antibodies were detected using SuperSignal West Dura (Pierce), and images collected using a BioRad Chemidoc and Image Lab software.