FIGURE 3.

ZFP91 stabilizes NIK and induces Lys⁶³-linked NIK ubiquitination. A, HEK293 cells were co-transfected with pNF-κB-Luc and Myc-NIK in combination with HA-Ub or FLAG-ZFP91 or together with HA-Ub and FLAG-ZFP91. Dual luciferase activity was measured 24 h after transfection. Data are represented as mean ± S.D. of three independent experiments. B, the lysates from HEK293 cells co-transfected with HA-Ub, FLAG-ZFP91, or both were subject to endogenous NIK and p52 immunoblots (top two panels). FLAG and tubulin immunoblots were also completed from the same cell lysates (bottom two panels). C, the lysates of HEK293 cells co-transfected with Myc-NIK, FLAG-ZFP91, and HA-Ub constructs as indicated were subject to immunoprecipitation (IP) using anti-Myc antibody, and the immune complexes were analyzed by immunoblots (IB; top two panels). FLAG, Myc, and tubulin immunoblot was completed from the same cell lysates (bottom panel). D, the lysates of HEK293 cells co-transfected with Myc-NIK and HA-UbK48R along with a control siRNA, ZFP91 siRNA, or FLAG-ZFP91 were incubated with anti-Myc antibody, and the immune complexes were analyzed by immunoblots (top two panels). The immunoblots using anti-ZFP91, anti-Myc, and anti-tubulin antibodies were completed from the same cell lysates (bottom two panels). E, the lysates of HEK293 cells co-transfected with Myc-NIK, FLAG-ZFP91, and HA-Ub constructs as indicated were subject to immunoprecipitation using anti-Myc antibody, a portion of the precipitated pellets were saved for immunoblots, and the remainder of the precipitated pellets were treated with 1% SDS, PBS to disrupt non-covalent interactions, and supernatants were diluted with lysis buffer, followed by a second immunoprecipitation using anti-Myc antibody. The immune complexes were analyzed by immunoblots (top two panels). FLAG immunoblot was completed from the same cell lysates.