Figure 1

Caspase-8 (Y380E) mutant mimics constitutive phosphorylation. (a) Primary-amino acid sequence of caspase-8 catalytic subunits indicating the putative phosphorylation site present in the linker region in red. Mach-α1 (Caspase-8a) numbering is used throughout. (b) (i) SH-SY5Y cells expressing caspase-8 with Tyr (WT), Glu (Y380E) or Phe (Y380F) at amino acid 380 were immunoprecipitated with an anti-caspase-8 antibody and protein A/G beads and immunoprecipitated samples analysed by SDS–PAGE and immunoblotted for p85α, total caspase-8 or phospho-Tyr380-casp-8b. Wild-type caspase-8 phosphorylation was induced by a 30 min treatment with EGF (25 ng/ml). (ii) Protein expression levels of caspase-8 in SH-SY5Y cells expressing caspase-8 WT, Y380E or Y380F variants show equal expression across the reconstituted cell lines. (c) SH-SY5Y cells overexpressing caspase-8 were grown to confluence on a fibronectin substrate, the monolayer wounded and the percentage migration into the wound measured after 24 h. Data shown are the mean+s.e.m. of n=3. The effect of Src inhibition was investigated by 16 h pre-treatment with Src-Inhibitor-1 (5 μm). *P<0.05; n.s. denotes not statistically significant. (d) Wild-type Jurkat cells (clone A3) or SH-SY5Y cells overexpressing caspase-8b variants were incubated with TNFα (50 ng/ml) for 6 h and quantitative RT–PCR expression analysis of the NFκB target gene IL-6 measured relative to Ribosomal Protein L18. The fold-change in TNF-treated cells relative to untreated matched control cells was calculated using the ΔΔCt method. Data shown are mean of triplicate repeats+upper range. No significant difference was observed between the SH-SY5Y cell lines. Jurkat cells served as a positive control. (e) Wild-type-Jurkat cells or SH-SY5Y cells overexpressing caspase-8b variants were incubated with TNFα (50 ng/ml) for up to 30 min and cells harvested at the time points indicated. Levels of caspase-8, total and phosphorylated IκB and tubulin were assessed by immunoblotting.