FIG. 4.

RE-IIBP represses transcription and associates with HDAC. (A) HeLa cells were transfected with Gal4-SV40 and increasing concentrations of RE-IIBP constructs, as indicated. Following transfection of the pcDNA3.1-HisTOPO-RE-IIBP and the point mutant (C483A), cell extracts were assayed for luciferase activity. (B) Cotransfections of a Gal4-SV40 luciferase reporter with the GAL4 DNA binding domain alone or Gal4-RE-IIBP and Gal4-RE-IIBP point mutant (C483A) are shown. (C) HeLa cells were transfected with Gal4-SV40 reporter vector, pcDNA3.1-HisTOPO-RE-IIBP, and CMX-HDAC1. TSA was added, as indicated, 24 h after transfection. Expression of RE-IIBP and HDAC1 used in transfection assays was confirmed by immunoblotting using anti-His and anti-HDAC1 antibodies. (D) GST-RE-IIBP and GST were incubated in HeLa cell extract and immunoblotted with anti-HDAC1 and anti-HDAC2 antibodies (I). HeLa cells were transfected with pcDNA3.1-HisTOPO-RE-IIBP (RE-IIBP) and pcDNA3.0 (pcDNA) constructs as indicated (II). After cell lysates were treated with DNase, immunoprecipitation was performed with anti-HDAC1, anti-HDAC2, anti-His, and anti-IgG antibodies; immunoblot analysis was then performed with anti-His (RE-IIBP) or anti-HDAC1 or anti-HDAC2 antibodies. (III) CMX-HDAC1 was in vitro transcribed and translated and incubated with purified GST-RE-IIBP and GST bound to beads. Pulled-down proteins were analyzed by SDS-PAGE and phosphorimager. IP, immunoprecipitation; IB, immunoblotting; WB, Western blotting.