Figure 1.

Generation and expression of sTRAIL specific variants. (A) Table showing the mutations in sTRAIL^wt leading to sTRAIL^DR5 (TRAIL-R2 specific) and sTRAIL^DR4 (TRAIL-R1 specific) variants. The TRAIL receptor specific variants were generated by site-directed mutagenesis inserting a D269H amino acid change (sTRAIL^DR5) and an amino acid change (S159R) to generate sTRAIL^DR4 in the sTRAIL ectodomain. (B) All 3 sTRAIL expression constructs were transiently transfected into 293 cells. After 24 h whole cell lysates were analyzed by Western blotting for TRAIL revealing similar expression levels for all 3 constructs. As control, expression from a conventional construct encoding membrane-bound, full-length TRAIL (FL) without hFIB, Furin CS or ILZ segments was also analyzed. EGFP transfected cells (ctrl) served as additional control. CuZnSOD was used as a loading control (C): Results of ELISA analyses for TRAIL showing the levels of secreted sTRAIL^wt (brown), sTRAIL^DR5 (red) and sTRAIL^DR4 (green) into the supernatant of transfected 293 cells. Results for cells transfected with the EGFP control plasmid (ctrl; gray) are also shown.