Engagement of xTRAIL1 to xDR-Ms. (A) 293T cells were transfected with a pEF1 expression plasmid for xDR-M1-Myc-His, xDR-M2-Myc-His, DR4-Myc-His, DR5-Myc-His, or with empty vector. Twenty-four hours after transfection, cell lysates from 10⁵ cells prepared, and the expressions of DRs were detected using one-twentieths of the cell lysate by Western blotting (WB) with an anti-Myc antibody. GST-FLAG and GST-FLAG-xTRAIL1 were expressed in E coli, purified, separated by SDS-PAGE, and detected by Coomassie brilliant blue (CBB) staining. Five micrograms of GST-FLAG or GST-FLAG-xTRAIL1, which was immobilized on glutathione-agarose beads, was mixed with the remaining cell lysate containing each of the DR-Myc-His. The protein complexes were washed extensively, and the precipitates were separated by SDS-PAGE and analyzed by Western blotting with an anti-Myc antibody. The relative binding activities are given as the fold increase over the value obtained with xDR-M1. (B) 293T cells were transfected with a pEF1 expression plasmid for DR (left panel, xDR-M1-Myc-His, xDR-M2-Myc-His, DR4-Myc-His, or DR5-Myc-His [10 or 30 ng]; right panel, xDR-M1-Myc-His, xDR-M1-2-Myc-His, or xDR-M2-Myc-His [2 or 10 ng]), a pNF-κB-luc reporter plasmid (200 ng), and pRL-TK-luc (50 ng) as a control for transfection efficiency. After 10 h, the cells were treated with cross-linked FLAG-xTRAIL1 (100 ng/mL). Twenty-four hours after transfection, the luciferase activity was measured. Firefly luciferase activity was normalized to the Renilla luciferase activity. Each relative luciferase activity is shown as the fold increase compared with the value obtained with pEF1 empty vector. The data represent the mean and SD from 3 separate experiments; *P < .01 compared with control. LBR indicates ligand-binding region; and DD, death domain.