Figure 1.

TNF treatment induces activation of AMPK. (A) L929 cells were transfected with non-targeting (siCtrl) or Ripk3 siRNAs (siRipk3). 48 h post transfection, cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for the indicated times. Then, whole cell lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. A compilation of representative immunoblots is shown; three ACTB immunblots are shown, but each protein was normalized to its corresponding loading control. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (scr, 0 h TQ). Results are mean + SD from at least 3 independent experiments. Statistical analysis was done by repeated measures two-way ANOVA (corrected by Sidak’s multiple comparisons test between siRNAs and corrected by Tukey’s multiple comparisons test between time points). Statistically significant differences within non-targeting siRNA-transfected cells (compared to scr, 0 h TQ) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001. (B) Ripk3 WT and KO MEFs were exposed to indicated treatments (medium [M], 30 ng/ml TNF [T], 100 nM SMAC-mimetic [S], 20 µM z-VAD [Z]) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins