The overexpression of NIK transforms rat fibroblasts in an NF-κB–dependent manner. (A) Top 2 panels: 5 μg of nuclear extracts prepared from B5 and h12 cells transduced with empty vector (EV2) or SR-IκBα (SR) were analyzed by EMSA, using NF-κB and Oct-1 probes. Middle 5 panels: whole-cell extracts (30 μg) of B5 or h12 infectants were subjected to SDS-PAGE and immunoblotting with anti-p52, antiphospho-IκBα, anti-IκBα, or antiactin antibodies. Bottom panel: HA-tagged NIK was immunoprecipitated from B5 and h12 infectants with anti-HA antibody and detected by immunoblotting with anti-NIK antibody (H-248). (B) Phase-contrast micrographs of cells cultured on monolayers (top images) or in soft agar (bottom images). B5 or h12 cell clones expressing wild-type NIK (NIK#1 and NIK#2) or not (EV1) were cultured in soft agar for 3 weeks. These cells were further transduced with SR-IκBα, and then pooled cells were assayed for anchorage-independent growth in soft agar. B5 and h12 cell clones expressing kd-NIK were also examined. Original magnification ×100. Scale bar represents 100 μm. SR indicates super-repressor; kd-NIK, catalytically inactive NIK; IB, immunoblotting; IP, immunoprecipitation.