Expression of DR5 and Fas in DcR3.Fc-treated DCs. (A) Expression of DR5 and FAS in DCs was determined by quantitative RT-PCR. Levels of the housekeeping gene GAPDH mRNA were used as internal controls for normalization of RNA quantity and quality differences in all samples. *P < .05 compared with nontreated group. (B) Surface expression of DR5 and Fas by flow cytometry. Cells were incubated with PE-conjugated anti-DR5 or PE-conjugated anti-Fas mAb before FACScan analysis. Shaded histograms indicate isotype control Abs; open histograms, specific Abs. (C) Time course of the formation of DR5 DISC. Monocytes (3 × 10⁷) were incubated with GM-CSF (200 ng/mL) and IL-4 (10 ng/mL) in the presence of 10 μg/mL Fc-fusion proteins or IgG₁ for different time intervals. Cells were lysed, and the assembled DISCs were immunoprecipitated with Sepharose beads adsorbed with anti-DR5 antibody and analyzed by Western blotting using antibodies to FADD and caspase-8. (D) Effect of MAPK inhibitors on the expression of DR5 and Fas. Monocytes (5 × 10⁵) were pretreated with kinase inhibitors for 1 hour before incubation with GM-CSF (200 ng/mL) and IL-4 (10 ng/mL) in the presence of 10 μg/mL Fc-fusion proteins or IgG₁, for 12 hours and 72 hours. Shaded histograms indicates isotype control Abs; open histograms, specific Abs. Error bars represent SD of the mean.