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. Author manuscript; available in PMC: 2014 Aug 28.
Published in final edited form as: Nat Commun. 2014 Feb 28;5:3350. doi: 10.1038/ncomms4350

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

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 1h 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 H9G mutant FLIP(S). Cells were treated with 2ng/mL rTRAIL for 16 hours; mean values and standard deviations 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 H9G mutant FLIP(S). Cells were treated with indicated concentrations of rTRAIL for 16h; mean values and standard deviations 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 1h 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 24h prior to treatment. The DR5 DISC was captured 1h 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 1h 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 10μM z-VAD-fmk for 1 hour prior to treatment with 5ng/mL rTRAIL for a further hour. Immunoprecipitates were analysed for the presence of Flag-tagged proteins. * denotes p<0.05 and ns denotes non-significant compared to EV control as determined by t-test.