Figure 2

Recombinant Caspase 8 Y380E interacts with endogenous p85α. (a) CD95-IcD pull-downs from Jurkat caspase-8 null lysates using purified recombinant Casp-8b (WT, C360A or non-cleavable quadruple mutant (Quad)). GST beads alone were used as a control to rule out non-specific binding. Beads and supernatant were immunoblotted for FADD, caspase-8 and active caspase-3 subunits (Asp175). Note that caspase-3 was not detected in the Bead fraction post-incubation. (b) Lysates from Jurkat caspase-8 null cells reconstituted with purified recombinant caspase-8 with Tyr (WT), Glu or Phe at amino acid 380 were immunoprecipitated with anti-caspase-8 antibody and protein A/G beads. Inputs and Immunoprecipitated samples were analysed by SDS–PAGE and immunoblotted for p85α, total caspase-8 or phospho-Tyr380-casp-8. (c) Recombinant caspase-8 WT, Y380E or Y380F was incubated in the presence or absence of Src and zVAD-FMK (100 μm) for 3 h at 37 °C and subsequently subjected to SDS–PAGE and immunoblotted for total caspase-8 or phosphorylated tyrosine residues.