RIP140 physically interacts with RelA. (A) Coimmunoprecipitation of HEK293T cells transfected with FlagRIP140 or an empty vector using anti-FLAG M2 antibody. Bound proteins were resolved by SDS-PAGE and subsequently detected by Western blot using RelA and FLAG M2 antibodies. Cells were treated with TNFα (1 μg/mL) for 1 hour before harvesting. (B) Mammalian-2-hybrid assay in HEK293T (left) or RAW264.7 (right) cells using a GAL4-Luc reporter cotransfected with Gal4-DNA–binding domain (DBD)-RelA or the empty Gal4-DBD and VP16-RIP140 or VP16 alone as indicated. Data are means plus or minus SEM (n = 9). *P < .05. (C,D) Pulldown assays performed with full-length RelA, the transactivation domain (TA), or the Rel-homology domain (RHD) of RelA fused to GST and GST alone as a control. GST fusion proteins 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 representations of RIP140-RelA interactions shown.