FIG 1.

MC159 inhibits NEMO polyubiquitination. (A) HEK 293T cells were transfected as follows: 1,000 ng pCI (lane 1), 500 ng pCI and 500 ng pUb-Myc (lane 2), 500 ng pCI and 500 ng pNEMO-FLAG (lane 3), 250 ng pNEMO-FLAG and 750 ng pUb-Myc (lane 4), 500 ng pNEMO-FLAG and 500 ng pUb-Myc (lane 5), and 750 ng pNEMO-FLAG and 250 ng pUb-Myc (lane 6). At 24 h posttransfection, cells were lysed in Ub lysis buffer. (B) HEK 293T cells were cotransfected with 500 ng pNEMO-FLAG, 500 ng pUb-Myc, and 1,000 ng of either pCI, pMC159, or pNEMOΔZnF. At 24 h posttransfection, cells were lysed in Ub lysis buffer. (C) HEK 293T cells were transfected with 250 ng pNEMO-FLAG, 250 ng pUb-Myc, and 1,000 ng of either pCI, pMC159, or pNEMOΔZnF. At 24 h posttransfection, cells were incubated in medium containing 10 ng/ml TNF for 15 min prior to lysis in Ub lysis buffer. For all experiments, a portion of each lysate prior to immunoprecipitation was set aside to detect protein expression by IB. The remaining cellular lysates were immunoprecipitated with anti-FLAG antibody or an IgG isotype control antibody 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 polyubiquitinated NEMO, MC159, or actin. Monoubiquitinated (Mono-Ub) and polyubiquitinated (Poly-Ub) forms of NEMO are indicated.