RIP140 associates with CBP in vivo. (A) Coimmunoprecipitation of HEK293T cells transfected with FlagRIP140 or an empty Flag vector using anti-FLAG M2 antibody. Bound proteins were resolved by SDS-PAGE and subsequently detected by Western blot using CBP and FLAG M2 antibodies. Cells were treated with TNFα (1 μg/mL) for 1 hour before harvesting. (B) Mammalian-2-hybrid assay in HEK293T cells using a GAL4-Luc reporter cotransfected with Gal4-DBD-CBP or the empty Gal4-DBD and VP16-RIP140 or the empty VP16 alone as indicated. Data are means plus or minus SEM (n = 9). *P < .05. (C,D) Pulldown assays performed with different CBP deletion mutants (as indicated) fused to GST and GST alone used as a control. GST deletion constructs were incubated with in vitro translated full-length RIP140 (C) or in vitro translated repression domains 1 to 4 (RD1-4) of RIP140 (D). Bound proteins were resolved by SDS-PAGE and visualized by autoradiography. Input lanes represent 10% of the input. Schematic representation of RIP140/CBP interactions shown.