CYLD regulates RIP1 ubiquitination in the necrosome.

(A-B) Increased RIP1 ubiquitination in the absence of CYLD. (A) CYLD^+/+ and CYLD^-/- MEFs or (B) control and CYLD knock-down HT29 cells were treated with TNF, the Smac mimetic LBW242 [35] and zVAD-fmk for the indicated times. Total lysis in 1% SDS was performed as described in the methods. The level of RIP1 ubiquitination was examined by Western Blot. NS: non-specific band. (C) Necrosome-associated RIP1 contains ubiquitin chains of different linkage types. Cells were treated with TNF and zVAD-fmk for 3 hours or left untreated. RIP3 immune complexes were denatured in urea, followed by immunoprecipitation with the indicated antibodies against ubiquitin. (D) Selective accumulation of poly-ubiquitinated proteins in RIP1 complexes in the NP-40 insoluble compartment. WT MEFs or CYLD^-/- MEFs were treated to undergo necrosis with TNF, zVAD-fmk, and CHX for the indicated times. Cells were lysed in NP-40 lysis buffer and insoluble material was solubilized with SDS. RIP1 was immunoprecipitated from both fractions followed by Western blot with the indicated antibodies.