ARC suppresses the enzymatic activity of caspase-8 in intact cells. (A) 293T cells were cotransfected with 0.2 μg of pcDNA3-caspase-8-AU1 or pcDNA3-caspase-8-mut and 2 μg of pcDNA3-ARC-Flag or pcDNA3. Caspase-8 in cell extracts was immunoprecipitated with anti-AU1 antibody and immunoprecipitates were incubated with the fluorogenic substrate acetyl-Asp-Glu-Val-Asp7-amino-4-methylcoumarin. ○, pcDNA3 alone; •, pcDNA3-ARC-HA alone; □, pcDNA3-caspase-8-AU1 alone; ▪, pcDNA3-caspase-8-AU1 and pcDNA3-ARC-HA; ▵, pcDNA3-caspase-8-mt-AU1 alone. (B) AU1-tagged caspase-8 and caspase-8-mt were detected in immunoprecipitates with anti-AU1 by immunoblotting.