Figure 1: Assessment of DR5 DISC stoichiometry.
(a) Quantitative western blot analysis of FLIP, FADD and caspase 8 levels in DR5 DISC IP samples was carried out in A549, H460 and HCT116 cells seeded at 5 × 106 and exposed the next day to equal amounts of the agonistic anti-DR5 antibody crosslinked to magnetic beads for 30 min. For each protein, 20% of the DISC IP sample was assessed alongside serial dilutions of each recombinant protein: His-tagged FLIP(S); untagged FADD and p18-caspase 8. (b) Bands were detected and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences). Standard curves were generated for each recombinant protein. These were second-order polynomials, from which the absolute amounts of FLIP, FADD and caspase 8 recruited to the DISC were calculated. (c) Femtomoles (fmol) of FLIP, FADD and caspase 8 recruited to the DR5 DISC in A549, H460 and HCT116 cells. For caspase 8, the levels of p53/55 and p41/p43-caspase 8 were added together. For FLIP, p43-FLIP(L) and FLIP(S) were added together. The result of a representative experiment is presented. (d) Ratios of the fmol of FLIP to FADD and caspase 8, caspase 8 to FADD and FLIP/caspase 8 to FADD recruited to the DR5 DISC in A549, H460 and HCT116 cells. The mean ratios and s.d. from three independent experiments are shown. (e) Ratios of the amount of FLIP to FADD and caspase 8, caspase 8 to FADD and FLIP/caspase 8 to FADD recruited to the DR5 DISC in A549 cells treated for 15, 30 and 60 min with anti-DR5-conjugated magnetic beads. The mean ratios and s.d. from three independent experiments are shown. (f) The ratios of DED-containing proteins to one another were calculated from DR5 DISC IP experiments using sub-lethal amounts of anti-DR5-conjugated magnetic beads. In these experiments, A549 cells were treated with 25% the number of beads used in other experiments. The mean ratios and standard deviations from three independent experiments are shown.
