Figure 1
HECTD3 interacts with caspase-8 through the DOC and DED domains. (a) Schematic representation of the HECTD3 and caspase-8 proteins and their mutants. (b) WT HECTD3 interacts with endogenous caspase-8, but not caspase-3 and -7. Flag-HECTD3, Flag-HΔ1–511 and an empty vector were transfected into HEK293T for 2 days. Immunoprecipitation was performed using the anti-FLAG M2 beads. (c) Caspase-8 interacts with HECTD3. Flag-caspase-8 or an empty vector was co-transfected with HECTD3 into HEK293T for 2 days. Immunoprecipitation was performed using the anti-FLAG M2 beads. (d) HECTD3 directly binds to caspase-8 but not caspase-3 in vitro. The recombinant GST-HECTD3 and GST were purified from E.coli (Supplementary Figure S1A) and were incubated with in vitro-translated caspase-8 and caspase-3 proteins, respectively, with Glutathione-Sepharose 4B slurry beads overnight at 4 °C. The beads were washed three times with 1 ml of 1 × cell lysis buffer. The proteins were resuspended with 20–50 μl of SDS sample buffer and analyzed by WB. (e) The endogenous HECTD3 protein forms a complex with the endogenous caspase-8 protein in HeLa. The endogenous HECTD3 was immunoprecipitated by the anti-HECTD3 Ab. Rabbit IgG was used as a negative control. (f) Both HECTD3 and caspase-8 are predominantly localized in the cytoplasm. The localization of Flag-HECTD3 and caspase-8 proteins was examined by immunofluorescence staining in HEK293T cells after co-transfection. DAPI was used to stain cell nuclei. (g) HΔ1–511 and HΔ109–393 significantly decrease the binding affinity with caspase-8. Flag-HECTD3 and its mutants were co-transfected with caspase-8 into HEK293T for 2 days. Immunoprecipitation was performed using the anti-FLAG M2 beads. (h) GST-fused H109–393 is sufficient to bind to caspase-8 as determined by performing the GST pull-down assay. (i) Flag-HECTD3 binds to both DED1 and DED2 of caspase-8 as determined by the GST pull-down assay
