(A) Immunoblot analysis of caspase activation, in SW480 cells expressing either ezrin S66A, S66D, R579A or WT, 16 or 6 hours after His-TRAIL (20 or 200 ng/ml) stimulation, respectively. (B) Analysis of TRAIL DISC formation in SW480 cells expressing either S66A, S66D, or WT ezrin. Cells were stimulated with 5 μg/ml Flag-TRAIL cross-linked with 10 μg/ml anti-Flag (M2) antibody. After cell lysis, the DISC was immunoprecipitated and analyzed by western blot. (C) Analysis of Bax activation. Ezrin S66A, S66D, WT and Mock-infected SW480 cells were left untreated or stimulated with His-TRAIL (20 or 200 ng/ml) for 16 h, then permeabilized and stained with an antibody recognizing active Bax before analysis by flow cytometry. The effect of two different concentrations of TRAIL (20 or 200 ng/ml, gray and black lines, respectively) were compared to unstimulated cells (gray filled curve). The percentage of cells containing active Bax after TRAIL stimulation is shown (upper and lower value, respectively).