Supplementary Data Supplementary Materials and Methods
نویسنده
چکیده
Plasmids and antibodies cDNA encoding AR from pSG5-AR was cloned into pSG5.HA, pSG5.FLAG, and pGEX-4T1, and cDNA encoding GATA2 (Open Biosystems) was cloned into pSG5.HA and pGEX-4T1. To generate a lentiviral vector expressing luciferase, cDNA encoding LUC2P from pGL4.35 (Promega) was cloned into pHR.CMV.FLAG.IRES-Hygro (1). cDNAs encoding CCAR1 fragments and AR fragments were cloned into pSG5.HA. The following plasmids were described previously: pSG5.HA-CCAR1, pSG5.FLAG-CCAR1, pGEX-5X-1-CCAR1, pSG5.HA-DBC1, MMTV-LUC, pHR.CMV.puro.Sin8-shNS, and pHR.CMV.puro.Sin8shCCAR1 (1,2). The lentiviral pLKO.1-puro vector (pLKO.1-shCCAR1 M1) containing the shRNA against CCAR1 (TRCN0000056005) was purchased from Sigma-Aldrich. Lentiviral particles were generated as described previously (2). PSA-LUC (pGL3_PSA540) and Probasin-LUC (ARR3-tk-luc/tk81-PB3) reporters were kind gifts from Gerhard Coetzee (University of Southern California). PSAGATA-LUC (lacking GATA binding motif) was generated with the QuikChange site-directed mutagenesis kit (Stratagene). The following antibodies were used in this study: anti-AR antibody N-20, anti-CCAR1 antibody C-20, antiDBC1 antibody H2, anti-MED1 antibody C-19, anti-RNA polymerase II antibody 8WG16, anti-GATA2 antibody H-116, and anti-tubulin antibody TU-02 (Santa Cruz Biotechnology); anti-FLAG monoclonal antibody (M2) and anti-FLAG M2 agarose (Sigma); anti-HA antibody 3F10 (Roche); anti-CCAR1 antibody 435A and anti-DBC1 antibody 434A (Bethyl Laboratories); anti-acetyl histone H3 antibody 06-599 (Millipore); anti-PSA antibody A0562 (Dako); anti-TMPRSS2 antibody 3209-1 (Epitomics); anti-luciferase antibody C-12 (Santa Cruz Biotechnology).
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