Role of Fas in CD40-mediated apoptosis of mature DCs. (A) Fas redistribution into lipid rafts following CD40 activation of mature DCs. Immature or LPS-matured DCs were activated for 1 hour with 5 μg/mL anti-CD40 (BB20) or isotype control, then washed and stained with anti-Fas-PE and Alexa 488-conjugated cholera toxin B (CTxB) before confocal microscopy. CTxB staining, Fas staining, overlay of both, and colocalization analysis are shown for each image. The white bar in the bottom right corner corresponds to 10 μm. (B) FAS-FADD coimmunoprecipitation. LPS-matured DCs were activated for 1 hour with anti-CD40 before Fas immunoprecipitation. Complexes were resolved on SDS-PAGE and membranes were probed with anti-FADD antibody then reblotted with anti-Fas as reported in “Materials and methods.” For each experiment shown in this figure, similar results were obtained in 2 independent experiments. (C) Effect of Fas-specific siRNA on CD40-mediated apoptosis. TNF-α-matured DCs (see “Materials and methods”) were transfected with Fas-specific siRNA and stained 18 hours later for Fas and CD86 expression. Dashed line indicates isotype control IgG1-PE staining of siRNA-transfected DCs; thin line, isotype control IgG1-PE staining of mock-transfected DCs; thick line, Fas siRNA- or irrelevant siRNA-transfected cells; filled histogram, mock-transfected cells. Transfected cells were then incubated for 6 hours with 2 μg/mL anti-CD40 (BB20) or isotype control and stained with annexin V FITC and 7-AAD, as previously described. Results were expressed as the percent inhibition of anti-CD40-induced apoptosis in DCs transfected with Fas-specific siRNA or irrelevant siRNA, as compared to mock-transfected cells; that is, 100 - [[(% apoptotic siRNA-transfected DCs + anti-CD40) - (% apoptotic siRNA-transfected DCs + isotype control)] × 100]/[(% apoptotic mock-transfected DCs + anti-CD40) - (% apoptotic mock-transfected DCs + isotype control)]]. Results are the means ± SEM of 2 independent experiments.