Fig. 6: rhTRAIL treatment results in association of RALB with the DR5-DISC (DR5-bound death-inducing signalling complex).

A Membrane (MF) and cytosolic (CF) fractions were isolated from HCT116 cells, and equal amounts of protein were immunoblotted for DR5, RALB, EGFR and MEK1/2. WCL = whole-cell lysate B Top: WB analysis of DR5-DISC IP performed in HCT116 cells. The DR5-DISC was captured magnetically after the addition of AMG 655-conjugated beads to cells for the indicated times. Co-immunoprecipitation of RALB, AP2α, FLIP, FADD and Caspase-8 was assessed and pull-down of DR5 confirmed. Protein expression in the non-DISC recruited fraction (unbound fraction) was also analysed. Bottom: HCT116 cells were transfected with either SC or siRALB for 24 h. A DR5-DISC IP was performed as described above, following a 1 h and 3 h incubation with AMG 655-conjugated beads. Co-immunoprecipitation of RALB, FLIP, FADD and Caspase-8 was assessed and pull-down of DR5 confirmed. Protein expression in the non-DISC recruited fraction (unbound fraction) was also analysed. C HCT116 cells were transiently transfected with empty vector (EV), FLAG-RALB WT and C-terminal Myc-tagged long isoform of DR5 (DR5_L) for 24 h followed by incubation with 0.5 ng/ml isoleucine zipper (iz)-TRAIL for 5 h. FLAG-RALB was immunoprecipitated using Anti-FLAG® M2 dynabeads and the expression of Myc-tag and FLAG-tag was determined by WB. Protein expression for Myc and FLAG were also determined by WB in the input lysates. D HCT116^Caspase-8+/+ and HCT116^{Caspase-8−/−} cells were incubated with AMG 655-conjugated dynabeads for 1 h and 3 h. The DR5-DISC was isolated and expression of RALB, Caspase-8, FLIP, FADD and DR5 was determined by WB. Protein expression in the unbound fraction was also analysed. E Top: HCT116 cells were transiently transfected for 24 h with 1 µg of EV, FLAG-RALB WT, FLAG-RALB G23V (constitutively active), FLAG-RALB S28N (dominant negative) or FLAG-RALB C203S (geranyl-geranylation deficient) for 24 h. The DR5-DISC was captured 3 h after the addition of AMG 655-conjugated beads to cells. Co-immunoprecipitation of FLAG-RALB was assessed and pull-down of DR5 was confirmed using WB analysis. Protein expression for FLAG in the unbound fraction was also analysed. Densitometry was performed on the DR5-DISC bound FLAG-RALB blot using ImageJ software and the results (intensity values) are denoted below the blot. Nd = not detected. Bottom: Apoptosis in the unbound fraction was determined using a Caspase-3/7 activity assay. F HCT116 cells were transiently transfected with EV or FLAG-RALB WT for 24 h, followed by treatment with rhTRAIL for 3 h. Expression of PARP, cleaved Caspase-3, FLAG-tag and DR5 was determined by WB (top). Apoptosis was determined using a Caspase-3/7 activity assay (bottom).