Phosphor rb

For immunoblotting, immunoprecipitation and immunostaining, we used antibodies against cyclin E1 (Santa Cruz, BioLegend or Millipore), cyclin D1, cyclin D3, cyclin B1, Cdk1, Cdk2, Cdk4, Cdk5, p107, Pcna, B-Myb, Fig 4, Rbbp9 (Santa Cruz), p130 (Santa Cruz or BD Transduction Labs or Bethyl Laboratories), HA (Covance), actin, Flag, Mybl1, cyclin A2 (Sigma), Gapdh (Invitrogen or Millipore), Lin9, Lin37, Lin52, Lin54, Rbbp4, Rbbp7 (Bethyl Laboratories), E2f4, Dp1, Cdc7 (Lab Vision), thiophosphate ester, Dmrtc2 (Abcam or Sigma), Mapk15 (Abcam), RalA, mTOR/Frap1, cyclin B3, phosphor-Rb, Tubulin and Miwi (Cell Signaling). Purified rabbit IgG was from either Bethyl Laboratories or Santa Cruz and mouse IgG from Santa Cruz. For immunoprecipitation, we also used anti-Flag M2 agarose (Sigma) and HA beads that were prepared by conjugating Protein A Sepharose (Amersham) with anti-HA antibody in 12CA5 ascites fluid (Covance).

Western blotting experiments were performed by following a previous study [25 (link)]. The primary antibodies for CXCR4 (Enzo Life Sciences, Farmingdale, NY, USA), phospho-p53, STAT3, phosphor-ERK1/2, ERK1/2, phosphor-Rb (Cell Signaling Technology, Danver, MA, USA), p53, p21, phosphor-STAT3, Nibrin (NBS1), breast cancer type 1 susceptibility protein (BRCA1), checkpoint kinase 1 (Chk1), Chk2, Cyclin A (Santa Cruz, CA, USA), zonula occludens-1 (Zo-1), occludin (OCLN) (Thermo Scientific, Waltham, MT, USA), and Claudin-5 (MILLPORE, Damstadt, DE, USA) were used. The concentration of antibodies used was 1:1000 for primary antibodies and 1:2000 for secondary antibodies. After reacting with the antibody, the protein was observed by chemiluminescence using Amersham Imager 600 (GE Healthcare, Chicago, IL, USA); beta-actin was used as the loading control.