Supporting Figures

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Fig. 7. The TR-Fc proteins exhibit similar stability and were recognized in their native conformation by receptor-specific antibodies. (A) Western blot analysis of the indicated TRAIL receptor (TR) fusion proteins that were either left untreated or kept in a 37°C water bath for 24 h. The proteins were resolved on nonreducing SDS/PAGE and detected by Western blot by using specific TR antibodies [TR1, AF347; TR2, AF837; TR3, MAB630; TR4, MAB633 (from R & D Systems)]. (B) The indicated fusion proteins were immunoprecipitated with the indicated antibodies for 2 h, washed, and resolved on SDS/PAGE. Retention of the fusion proteins was detected on Western blot by mouse anti-human IgG conjugated to horseradish peroxidase. The TRAMP-Fc fusion proteins were included as nonbinding controls.

Fig. 8. Flow cytometric analysis of expression of the transfected TRs. Jurkat cells transfected with the indicated yellow fluorescent protein (YFP)-tagged receptors were examined for YFP fluorescence or surface expression of hemagglutinin (for TR3). Only the YFP-positive cells were analyzed for response to TRAIL-induced apoptosis.

Fig. 9. The preligand assembly domain of dominant negative TR2, but not the ligand-binding domain, is required for inhibition of TRAIL-induced apoptosis in Jurkat cells. Jurkat cells transfected with the indicated plasmids were stimulated with TRAIL, and cell death was analyzed by flow cytometry for Annexin V staining.

Fig. 10. Expression of mutant TR4 on the cell surface. The indicated TR4 receptors were transfected into 293T cells and examined for surface expression by staining with an antibody against the hemagglutinin tag or a monoclonal antibody specific for TR4 (HS402) (bold curves). The shaded curves represent staining of cells transfected with TNFR-1/p60.