Figure 5.

Ablation of the RIP1 deubiquitinase CYLD prevents death signaling triggered by TAK1 inhibition. (a) Western blots for the indicated proteins from MEFs stably expressing CYLD shRNA (shCYLD) or a scrambled sequence (shScram) by lentiviral vectors, treated with 5z-7 plus TNFα or CHX plus TNFα, with or without zVAD for 4 h. (b) Western blots for RIP1 and RIP3 after IP with an anti-RIP3 antibody from extracts of shScram or shCYLD MEFs treated as indicated for 1 h. *indicates mobility shift. (c) Caspase 8 activity in shScram and shCYLD MEFs treated with vehicle control or 5z-7 plus TNFα in the presence or absence of Nec-1 or zVAD for 2 h. ^†P<0.01 versus Con; #P<0.05 versus corresponding shScram. (d) Cell death assessed by PI staining of cells treated as in c for 4 h. ^†P<0.01 versus Con; #P<0.05 versus corresponding shScram; ^§P<0.05 versus 5z-7 plus TNF