Figure 5
A20-deficient T cells are highly susceptible to apoptosis and necroptosis in vitro.
CD8+ T cells from CD4-Cre A20fl/fl and A20fl/fl control mice were isolated and stimulated with anti-CD3/CD28 and/or TNF, respectively, for the indicated time points. (a,b) Active caspase-3/7 was determined by flow cytometry using DEVD substrate after stimulation with (a) anti-CD3/CD28 only, or (b) anti-CD3/CD28 with TNF. (c) Dot Plots showing proliferation and caspase-3/7 activity after 72 h of stimulation. (d) Western blot analysis of full length caspase-8, cleaved caspase-8, full-length caspase-3 and cleaved caspase-3 after TCR or TNF stimulation, respectively. (e) Lysates from unstimulated and 48 h anti-CD3/CD28-stimulated CD8+ T cells were immunoprecipitated with anti-RIPK1. Protein concentrations were equalized by staining lysates for GAPDH. Beads plus lysate without antibody (B+L) and beads plus anti-RIPK1 without lysates (B+A) were used as controls. (f) CD8+ T cells were left untreated or stimulated with anti-CD3/CD28 for 72 h and IFC was performed. A representative of 2 independent experiments is shown, with 3 mice per group. Error bars indicate + SEM. Student’s t-test, *p < 0.05; **p < 0.01.
