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. Author manuscript; available in PMC: 2018 Aug 22.
Published in final edited form as: Circulation. 2017 Jun 1;136(8):729–742. doi: 10.1161/CIRCULATIONAHA.116.026240

Figure 4. Traf2 regulates necroptotic cell death signaling through TAK1 and TRADD.

Figure 4

A, Western blotting for the indicated proteins from cardiomyocytes infected with adenoviruses encoding TRADD shRNA (Ad-shTRADD) or a scrambled sequence (Ad-shScram) along with Adβgal or AdTraf2ΔR then treated with zVad plus TNF for 4 h. B, Cell death assessed by PI staining of cardiomyocytes treated as in A for 4 h. *P < 0.05 versus vehicle control; #P < 0.05 versus Adβgal zVad+TNF; §P < 0.05 versus shScram zVad+TNF in the corresponding group. C, Western blotting following IP with an anti-RIP3 antibody from extracts of cardiomyocytes treated as in A for 1 h. D, Western blotting for the indicated proteins from cardiomyocytes infected with Adβgal or AdTraf2ΔR along with an adenovirus expressing a constitutively active TAK1 mutant (AdTAK1ΔN), then treated with vehicle control or 10 ng/ml TNFa for 4 h with or without zVad. E, Cell death in cardiomyocytes treated as in D. *P < 0.05 versus vehicle control (Con); #P < 0.05 versus AdTraf2ΔR TNF; §P < 0.05 versus Ad-Traf2ΔR TNF+zVad. F, Western blotting following IP with an anti-RIP3 antibody (top) or pre-immune IgG control (bottom) from extracts of cardiomyocytes infected with Adβgal, AdTraf2ΔR, or AdTAK1ΔN, then treated with vehicle control or zVad plus TNFa for 1 h. One-way ANOVA with Bonferroni's post-hoc test was used in B and E. Data were from at least 3 independent experiments with ≥ 900 cells per group analyzed for cell death.