Figure 1
NIK interacts with CnAα/β through its kinase domain and C-terminal region.
A. Co-immunoprecipitation of CnAα (left) and CnAβ (right) with NIK and its mutants (ΔC, ΔKC, ΔN, ΔNK and ΔKC). NIK and its mutants expressed in cells are indicated at the top of panels. Control indicates the Flag-tagged expression vector. The upper panel (Co-IP) shows western blotting of immunoprecipitates using an anti-Flag antibody to detect co-immunoprecipitation of Myc-tagged CnAα or CnAβ. Band intensities of Co-IP bands relative to INPUT were normalized to that of full-length NIK and exhibited above the panel. The middle panel shows western blotting of total cell lysates using an anti-Myc antibody. The lower panels show western blotting of immunoprecipitates using the anti-Flag antibody to detect Flag-tagged NIK and mutants. Asterisks indicate bands of IgG chains used for immunoprecipitation. Results of one representative experiment of three are shown. Blots are cropped for clarity. Full-length blots of key data are presented in Supplementary Figure 2. B. Schematics of NIK and its deletion mutants used in this study. “Kinase” indicates the kinase domain. “IKKα” indicates the determined binding region of IKKα. A TRAF3-binding sequence is located in the N-terminal region. The Flag tag (abbreviated in this figure) was connected to the N-terminus of the wild-type protein and mutants. The binding ability of each protein for CnAα/β, as determined in Fig. 1A, is indicated at the right of each structure. “+” indicates positive for binding and “−” indicates negative for binding.
