Figure 4.

Ser-345 MLKL phopshorylation by RIPK3 is mediated by a RIPK1/RIPK3/MLKL complex. (a) KO, NEMO and NEMO-A323P cells were pre-incubated 1 h with the indicated compounds followed by TNF stimulation for the indicated time period (hour: h). Immunoblot analysis for the indicated proteins is shown. L.E., long exposure. (b) KO, NEMO and NEMO-A323P cells were pre-treated 1 h with zVAD followed by a time course (min) with 10 ng/ml TNF. Complex II was isolated by an FADD immunoprecipitation. Immunoblot analysis was performed to reveal the indicated proteins. (c) siCTR and siRIPK3 Nemo⁽⁻⁾ MEF cells were treated with TNF/zVAD as indicated. The MLKL complex was isolated by a first immunoprecipitation against MLKL (IP₁). The supernatants derived from IP₁ were subjected to a second IP against FADD (IP₂) to isolate the complex II. Ifmmunoblot analysis was performed to reveal the indicated proteins