Impaired RIP1 and RIP3 phosphorylation in the absence of CYLD.

(A) MEFs were treated with TNF, zVAD-fmk and the LBW242. Cell lysates were extracted by sequential detergent lysis in NP-40 and SDS as described in methods. RIP1 and RIP3 in each fraction were examined by Western blot. Note that phosphorylated RIP1 and RIP3 (p-RIP1 and p-RIP3) were exclusively detected in the SDS fractions. (B) HT-29 cells stably expressing non-specific (NS) shRNA or CYLD shRNA were treated with TNF, LBW242 and zVAD-fmk for the indicated times. Unmodified and phospho-RIP1 and phospho-RIP3 were analyzed by Western Blot.