Figure 4: Functional confirmation of the predominant orientation of the FLIP–FADD interaction. | Nature Communications

Figure 4: Functional confirmation of the predominant orientation of the FLIP–FADD interaction.

From: Differential affinity of FLIP and procaspase 8 for FADD’s DED binding surfaces regulates DISC assembly

Figure 4

(a) Western blot analysis of DR5 DISC IP carried out in HCT116 cells stably over-expressing wild-type and mutant FLIP(S). The DR5 DISC was captured 1 h after addition of anti-DR5-conjugated beads to cells. Co-immunoprecipitation of FLIP, FADD and caspase 8 was assessed and pull down of DR5 confirmed. (b) Annexin V/propidium iodide flow cytometry analysis of apoptosis in control HCT116 cells (EV) and HCT116 cells stably expressing wild-type (WT) or F114A or H7G mutant FLIP(S). Cells were treated with 2 ng ml−1 rTRAIL for 16 h; mean values and s.d. are shown (n=3). (c) Caspase 8-like (IETD-ase) and caspase 3/7-like (DEVD-ase) activity in control HCT116 cells (EV) and HCT116 cells stably expressing wild-type (WT) or F114A or H7G mutant FLIP(S). Cells were treated with indicated concentrations of rTRAIL for 16 h; mean values and s.d. are shown (n=3). (d) Western blot analysis of DR5 DISC IP carried out in H460 NSCLC cells stably over-expressing wild-type and mutant FLIP(S). The DR5 DISC was captured 1 h after addition of anti-DR5-conjugated beads to cells. Co-immunoprecipitation of FLIP, FADD and caspase 8 was assessed and pull down of DR5 confirmed. (e) Western blot analysis of DR5 DISC IP carried out in HCT116 cells transfected with wild-type and mutant Flag-FLIP(L) constructs. Cells were transfected for 24 h prior to treatment. The DR5 DISC was captured 1 h after addition of anti-DR5-conjugated beads to cells. Co-immunoprecipitation of Flag-tagged FLIP(L) and FADD was assessed and pull down of DR5 confirmed. (f) Western blot analysis of DR5 DISC IP carried out in HCT116 cells transfected with wild-type and mutant Flag-FLIP(S) constructs. The DR5 DISC was captured 1 h after addition of anti-DR5-conjugated magnetic beads to cells. Co-immunoprecipitation of Flag-tagged FLIP and endogenous FADD was assessed. (g) Western blot analysis of caspase 8 IP carried out in HCT116 colon cancer cells transfected with Flag-tagged wild-type or mutant FLIP(S) and FLIP(L) expression constructs. Twenty-four hours after transfection, cells were pre-treated with 10 μM z-VAD-fmk for 1 h prior to treatment with 5 ng ml−1 rTRAIL for a further hour. Immunoprecipitates were analysed for the presence of Flag-tagged proteins. *; ns, non-significant compared with EV control as determined by t-test.

Back to article page