Cyclin e2

Chemicals: Crystal Violet (Sigma), cycloheximde (Sigma), DMSO (Sigma), EdU (Invitrogen), entinostat (Selleckchem), Geneticin (Invitrogen), MG132 (Cayman), Noble Agar (Difco), puromycin (Sigma), trichostatin A (Cayman), tubastatin A (Cayman), tubacin (Selleckchem). Primary Antibodies: pH3Ser10 ab47297 (Abcam), p21/Kip1 610241 RB 554136 (BD), p53BP1ser25 A300-652A (Bethyl), cyclin D1 CC12 (Calbiochem), ac- α-Tubulin #5335, GAPDH #2118, HDAC6 #7612, PARP #9542, Ph-p53Ser15 #9284, pHistone H2A.X Ser139 #2577, pChk2Thr68 #2197, pERK1/2 Thr402/Tyr404 #4370, pRbSer780 #9307, pRbSer807/811#9308 (Cell Signaling Technology), FGFR3 sc-123, c-MYC sc-764, cyclin A sc-751, cyclin B1 sc-752, cyclin D2 sc-754, cyclin E sc-481, cyclin E2 sc-28351, ERK1 sc-94 (Santa Cruz Biotechnology), pATMSer1981 #05-740, ac-Histone H3 #06-599, ac-Histone H4 #06-866 (Upstate).

Lysates were prepared and electrophoresed on gels as described previously (26 (link)). Membranes were probed with primary antibodies following each of the supplier's recommendations. Cyclin A2, Cyclin B1, Cyclin D1, Cyclin E1, Cyclin E2, Cyclin H, CDK2, ERK2, and the secondary antibodies were purchased from Santa Cruz. The immunoblots were developed using ECL Western Blotting Substrate (Thermo Scientific) or Supersignal West Femto Chemiluminescent Substrate (Thermo Scientific).

RIPA lysis buffer (Millipore) was used to lyse cells in the presence of protease inhibitor cocktail (Roche). Protein concentration was measured using Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Same amounts of protein were resolved by 10% SDS-PAGE, followed by transfer to nitrocellulose membranes (GE Healthcare Life Sciences). PBST (0.1% Tween 20 in PBS) containing 5% skim milk was used to block the membranes. Immunoblotting was performed overnight at 4 °C with antibodies for OCT4 (Santa Cruz Biotechnology, sc-9081), cyclin A2 (Cell Signaling Technology, #4656), cyclin B2 (Santa Cruz Biotechnology, sc-28303), cyclin D1 (Millipore, 04–221), cyclin E2 (Santa Cruz Biotechnology, sc-28351), CDK4 (Cell Signaling Technology, 12790), CDK6 (Cell Signaling Technology, 3136), p21 (Cell Signaling Technology, 2947), β-catenin (Millipore, 04–958), Smad2/3 (R&D systems, AF3797), and β-actin (Sigma-Aldrich, A5441). The membranes were stained with secondary antibodies Alexa Flour 680-goat anti-mouse IgG (ThermoFisher Scientific) and IRDye 800CW-goat anti-rabbit IgG (LI-COR) for 1 h at room temperature. Protein bands were visualized with LI-COR Odyssey Imaging System (LI-COR).