Anti cyclin b

Cells were lysed on ice for 30 min using a lysis buffer (Thermo Fischer Scientific). The lysate supernatants were denatured, then separated on a 4–12% bis Tris gel (Invitrogen). Proteins in the gel were then transferred to a nitrocellulose membrane and probed with anti-phosphorylated (p)-ATM (Ser-1981, 1:500 dilution), anti-p-ATR (Ser-428, 1:500 dilution), anti-ATM (1:500 dilution), anti-ATR (1:1000 dilution), anti-p-p53 (Ser-392, 1:500 dilution), anti-p53 (1:500 dilution), anti-BRCA1 (1:500 dilution), anti-HIF-1α (1:250 dilution), anti-UBE2K (1:500 dilution), anti-cyclin B (1:1000 dilution), and anti-β-actin (1:1000 dilution) primary antibodies overnight at 4° C. All antibodies were purchased from Abcam. Membrane-bound primary antibodies were detected using appropriate secondary antibodies. Equal loading of protein was ensured by measuring β-actin expression.

Lysate harvesting and western blot analysis was performed as previously described [57 (link)]. To quantify the levels of keratin 10 (K10), insoluble cell pellets obtained from RIPA-SDS lysis were resuspended in 8 M urea, 10% β-mercaptoethanol, 2 mM PMSF, and incubated at room temperature while rotating for 30 min, as described previously [90 (link)]. The insoluble debris was removed by centrifugation at 14,000 rpm at 4’C. Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma). Secondary antibodies used were horseradish peroxidase (HRP)-conjugated anti-rabbit (Cell Signaling Technology) and HRP-conjugated anti-mouse (GE Life Sciences). Western blots were developed using Clarity Western ECL blotting substrate (Bio-Rad). Images were captured on either autoradiography film or Biorad ChemidocMP imaging system. Blots were analyzed with Biorad Imagelab 5.0 software.