Anti goat antibody

Cells were lysed in lysis buffer (100 mM Tris, pH 7.4, 1% sodium dodecyl sulfate (SDS), 10% glycerol), sonicated, and protein concentrations were assessed by the bicinchoninic acid (BCA) assay. Laemmli buffer (3×) was then added, and lysates were boiled for 5 min at 100°C. Cell lysates were separated with electrophoresis employing 8%-15% gels and then wet blotted to a nitrocellulose membrane (GE Healthcare, Amersham, #10600008, Little Chalfont, UK). Individual proteins were detected with specific antibodies: BRCA1 (Santa Cruz, sc-6954), CDK12 (Cell Signaling, #11973, Danvers, MA, USA), CDK13 (rabbit serum produced in-house), Cyclin K (Santa Cruz, sc-376371), p53 Pantropic Ab-6 (Millipore, OP43, Billerica, MA, USA), Cyclin T1 (Santa Cruz, sc-8127), CHK1 (Cell Signaling, #2360), CHK1-pSer296 (Cell Signaling, #2349), PARP (Cell Signaling, #9542), γH2AX pSer 139 (Biolegend, 613402, San Diego, CA, USA), p21 (Santa Cruz, sc-397), p27 (Santa Cruz, sc-528), pRb (Cell Signaling, #9309), and pRb-pSer780 (Cell Signaling, #8180). Anti-rabbit and anti-mouse secondary horseradish peroxidase (HRP)-linked antibodies were obtained from GE Healthcare (NA934V, NA931V), and anti-goat antibody was obtained from Sigma-Aldrich (A5420). The immunoreactive bands were visualized using the Western blot Luminol reagent (Santa Cruz Biotechnology, SC-2048).