NIK induces constitutive NF-κB activity in rat fibroblasts. (A) B5 and h12 cells were infected with retroviruses capable of expressing HA-tagged NIK (NIK) or catalytically inactive NIK (kd-NIK). Pools of B5 and h12 cells transduced with the control pMRX-HAiresPuro vector (EV1) were used as a control. Cytoplasmic extracts from EV1 and 2 independent cell clones (no. 1 and no. 2) were subjected to immunoprecipitation using antibody against the HA epitope. Immunoprecipitates were then resolved by 8% SDS-PAGE and subjected to immunoblotting with anti-NIK antibody. 293T cells were transiently transfected with the pMRX-HAiresPuro vector (EV1) or pMRX-HA-NIKiresPuro (NIK). Cytoplasmic extracts (30 μg) were then used for immunoblotting as negative and positive controls, respectively. (B) Elevated p52 production in rat fibroblasts. Whole-cell lysates from B5 and h12 cells expressing wild-type NIK or kd-NIK were subjected to SDS-PAGE and immunoblotting with anti-p52 for detection of p100 and p52 or antiactin antibodies. (C) Elevated NF-κB–DNA binding activity in rat fibroblasts; 5 μg of nuclear extracts prepared from B5 and h12 cells expressing wild-type NIK or kd-NIK were analyzed by EMSA, using oligonucleotides encoding an NF-κB–binding sequence or Oct-1–binding sequence as probes. (D) DNA-binding NF-κB components in B5 and h12 cells expressing wild-type NIK were analyzed by super-shift EMSA. Nuclear extracts (5 μg) from B5 NIK#1 and h12 NIK#2 cells were preincubated for 30 minutes with preimmune (PI), anti-p50, anti-RelA or anti-RelB sera, and then subjected to EMSA with the NF-κB–specific probe. IB indicates immunoblotting; IP, immunoprecipitation.