Fig. 4.
Cell death and survival of Ripk1−/−Ripk3−/−Tradd+/− and Ripk1−/−Ripk3−/−Tradd−/− cells. a Thymocytes were treated with indicated concentrations of TNFα for 16 h with 30 μg/mL cycloheximide. After addition of 1 μg/mL propidium iodide (PI), dead cells (PI+) were determined by flow cytometry (performed in triplicate, presented as mean ± SEM, n = 3 independent experiments). b Mature T cells were isolated from the lymph nodes and spleen, labeled with CellTrace Violet (Invitrogen), pretreated for 1 h with or without 50 μM zVAD-fmk, and stimulated with 5 μg/mL αCD3 and 1 μg/mL αCD28. At 48 h, cells were harvested, stained with PI, and analyzed by two-color flow cytometry (n = 3 independent experiments). c Western blot of cFLIP, Bcl-XL, and β-Actin in resting and stimulated mature T cells of indicated genotypes. Representative of three independent experiments. d, e Western blots of p65 phosphorylation, total p65, and β-Actin after stimulation of fibroblasts with d 10 ng/mL TNFα (n = 5 independent experiments) or e 1 μg/mL LPS (n = 3 independent experiments). Numbers under each gel indicate fold changes of signals for each lane