Effects of calpain inhibitors, colchicine, and FMF mutations on N330-induced IκB-α degradation. (A) HeLa cells were cotransfected with IκB-α–V5 and N330-myc, and treated with various inhibitors, ALLN (0.3-1.25 μg/mL, proteosome, calpain I and II inhibitor), Ro106-9920 (0.39-3.125 μM, ubiquitination inhibitor), calpain inhibitor III (0.625-5 μg/mL, calpain I and II inhibitor), EST (3.125-100 mM, calpain I inhibitor), or calpain inhibitor IV (1.25-5 μg/mL, calpain II inhibitor). Cells were also transfected with N330-myc or IκB-α–V5 alone as controls. Cell lysates were analyzed by Western blotting with anti-V5 Ab for IκB-α (top), antimyc Ab for N330 (middle), or anti–β-tubulin Ab for loading controls (bottom). The approximately 30-kDa IκB-α fragment is denoted with an asterisk. (B) HeLa cells were cotransfected or transfected as in panel A. Cotransfected cells were treated with various amounts of colchicine (5-80 pg/mL). Cell lysates were analyzed as in panel A. (C) PBMCs from 3 FMF patients who had at least one mutation in the B30.2 domain, as well as from 3 healthy controls, were lysed immediately after purification. Cell lysates were subjected to Western blotting with antipyrin (top panel) or anti–IκB-α Abs (bottom panel). Asterisks denote the approximately 30-kDa IκB-α fragment. (D) PBMCs from 3 FMF patients who have 2 mutations on B30.2 domains of both alleles, as well as from 3 healthy controls, were treated with IFN-γ for 24 hours. Cells were lysed and analyzed as in panel C.