FIG 6.

MC132 interacts with p65 via the RHD of p65. (A) MC132 and p65 interact. HEK293T cells were seeded at 4 × 10⁶ cells per 10-cm plate and transfected with 8 μg pCEP4-MC132 or a plasmid expressing control p38-Flag, and 24 h later, the cells were lysed and immunoprecipitated (IP) with anti-Flag beads. Immunoblots were probed with the indicated antibodies. Representative blots are shown (n = 4). (B) MC132 interacts with the p65 RHD. The schematic shows full-length p65(1–551) and the different truncations tested. HEK293T cells were seeded at 4 × 10⁶ cells per 10-cm plate and transfected with 4 μg pCEP4-MC132 together with 4 μg vector encoding full-length p65-HA or the indicated p65 truncation mutants. Cells were lysed the following day, immunoprecipitated with anti-Flag beads, and probed for Flag-tagged MC132 or p65-HA. Representative blots are shown (n = 4). (C) Truncation of MC132 abolishes interaction with p65 and inhibition of NF-κB. HEK293T cells were seeded at 4 × 10⁶ cells per ml and transfected the following day with 8 μg pCEP4-MC132 or the MC132 truncation mutants indicated in the schematic. Cells were lysed the following day, immunoprecipitated with anti-Flag beads, and probed along with lysate controls for Flag-tagged proteins and endogenous p65. Representative blots are shown (n = 4). (D) The effect of MC132 truncations on TRAF2-stimulated NF-κB activation was measured by a reporter gene assay as described in the legend to Fig. 1. Data are percentages of the stimulation activity for control cells and are means ± SD for triplicate samples from a representative experiment (n = 4). *, P < 0.001 compared to the control.