Fig. 4. Tankyrases limit complex 2 assembly.
(A) TNF-induced complex 2 immunoprecipitation using anti-FLAG M2 affinity beads. Cells were treated with TNF (10 ng/ml) + Smac mimetic (50 nM) + caspase inhibitor (5 μM; TSI) ± IWR-1 (5 μM) for 1.5 hours. (B) TNF-induced complex 2 immunoprecipitation using anti–cleaved caspase-8 antibody. Tnks2−/− MDFs expressing Dox-inducible shTNKS1 were pretreated with ± Dox (1 μg/ml) for 48 hours followed by TNF (100 ng/ml) + Smac mimetic (250 nM) + caspase inhibitor (5 μM; TSI) for 2 hours. In Tnks2−/− MDFs, we observed two anti-tankyrase bands, one at ~150 kDa and one just below. Our data suggest that they are TNKS1 specific but are not the result of caspase cleavage, and we therefore believe them to be an unreported isoform. (C) TNF-induced complex 2 immunoprecipitation using anti-RIPK1 antibody. Ripk1D325A/+ heterozygous MDFs were treated with TNF (50 ng/ml) + Smac mimetic (100 nM) ± IWR-1 (5 μM) for 2 hours. (D) Cell death monitored by time-lapse imaging (IncuCyte) of PI staining over 16 hours. Ripk1D325A/+ heterozygous MDFs were treated with TNF (50 ng/ml) + Smac mimetic (25 nM; TS) ± IWR-1 for 16 hours. % PI positive (dead) was obtained by normalizing PI count to cell confluency. Dashed line denotes % PI positive (dead) without IWR-1 treatment for reference. Results from two additional independent MDFs are shown in fig. S4C. Filled arrowheads alone indicate potential tankyrase species. Double bands around 150 kDa in anti-tankyrase blots indicate full-length TNKS1 (upper band, 150 kDa) and an undefined TNKS1 isoform (lower band). * indicate IgG chains.