FIG 6.

MC159 reduces cIAP1-mediated NF-κB activation and NEMO polyubiquitination. (A) HEK 293T cells were cotransfected for 24 h with 225 ng pNF-κBluc; 25 ng pRL-TK; 250 ng pUb-myc; 250 ng pNEMO-FLAG; 750 ng of pcDNA3.1 (vector control [VC]), pMC159, or pK13-FLAG; and increasing amounts of pcIAP1. Cells were lysed, and luciferase activities were quantified. Results are shown as the fold induction of luciferase activity relative to those of cells transfected with the empty vector. *, P < 0.05; **, P < 0.001 (compared to cells transfected with the empty vector). Immunoblot analysis of lysates was also performed. (B) HEK 293T cells were cotransfected with 250 ng pNEMO-HA, 250 ng pUb-Myc, 250 or 500 ng pcIAP1, and an amount of pCI sufficient to make 1,500 ng present in all reaction mixtures. (C) HEK 293T cells were cotransfected with 250 ng pNEMO-HA, 250 ng pUb-Myc, 250 ng pcIAP1, and 1,000 ng of either pCI or pMC159. (B and C) At 24 h posttransfection, cells were lysed in 150 μl of Ub lysis buffer. A portion of each lysate was set aside to monitor protein expression. For the remaining clarified cellular lysates, IP was performed by using the indicated antibodies conjugated to protein G-Sepharose beads. Immunoprecipitated samples or cellular lysates were separated by SDS-PAGE, and proteins were transferred onto PVDF membranes for IB. Membranes were probed with the indicated antibodies to detect cIAP1, NEMO, MC159, K13, or actin. Monoubiquitinated (Mono-Ub) and polyubiquitinated (Poly-Ub) forms of NEMO are indicated.