Interaction of N330 with IκB-α and the N330-induced proteolysis of IκB-α. (A) V5-tagged IκB-α was cotransfected into PT67 cells with myc-tagged pyrin, N330, or C330 or empty vector. Lysates were immunoprecipitated with antimyc Ab and analyzed as in Figure 4B. (B) Lysates from PBMCs of a healthy donor were immunoprecipitated with anti–IκB-α Ab or control IgG. Cell lysates and eluted proteins were analyzed by Western blotting with antipyrin (top) or anti–IκB-α Abs (bottom). Data are from a representative experiment from 3 separate donors. (C) HeLa cells were transfected with V5-tagged IκB-α (IκB-α–V5) alone (i,ii), or cotransfected with IκB-α–V5 and N330-myc (iii-vi). After 24 hours, IκB-α and N330 were stained with AlexaFluor 488–conjugated anti-V5 Ab (green) and AlexaFluor 568–conjugated antimyc Ab (red), respectively. Cells were counterstained with DAPI (blue) and visualized on a Leica DMR microscope. Expressed IκB-α is shown in panels i, ii (merged with nuclei), and iii, and N330 is in panel iv. Merged images for IκB-α and N330 without and with nuclei are in panels v and vi, respectively. (D) HeLa cells were cotransfected with IκB-α–V5 and myc-tagged pyrin variants or empty vector. Cell lysates were analyzed by Western blotting with anti-V5 (top) or antimyc Abs (bottom). (E) HeLa cells were cotransfected with IκB-α–V5 and increasing amount of N330-myc, and also transfected with empty vector or N330-myc alone. Cell lysates were analyzed by Western blotting with anti–IκB-α (top) or antimyc Abs (bottom). (F) PT67 cells were cotransfected with IκB-α–V5 and the 10-aa deleted N-terminal fragments depicted in Figure 4A. Lysates were immunoprecipitated with antimyc Ab and analyzed as in Figure 4B. (G) HeLa cells were cotransfected with IκB-α–V5 and N330-myc, N265-myc, or empty vector, and also transfected with N330-myc alone. Cell lysates were analyzed by Western blotting with anti-V5 (top) or antimyc Abs (bottom).