Figure 1.

FL-TRAIL and sTRAIL are expressed in HEK293 cells, but only sTRAIL is secreted into the supernatant. (a) Schematic depiction of full length, membrane bound TRAIL (FL) and soluble TRAIL (sT) expression cassettes including depiction of the localisation of the two TRAIL forms when expressed in cells. The full-length version is the TRAIL cDNA corresponding to aa1-aa281 containing a cytoplasmic part (C), transmembrane region (TM) and the extracellular domain. The sTRAIL construct consists of a hFIB heterologous signal peptide, a Furin cleavage site (Furin CS), an Isoleucine Zipper (ILZ) and the sTRAIL part from aa114–281. Both constructs are under the control of the CMV promoter within pcDNA3 expression plasmids or adenoviral vectors. (b) HEK293 cells were transfected with pCDNA3 constructs for EGFP, FL-TRAIL (FL) or sTRAIL (sT). The resulting protein lysates were western blotted and probed with a TRAIL antibody. (c) HEK293 cells were transfected with an empty pCDNA3 plasmid (ctrl), as well as constructs for FL-TRAIL (FL) and secreted TRAIL (sT), respectively. The cells were then stained with a TRAIL antibody followed by a secondary antibody carrying a PE fluorescent tag and analysed by flow cytometry. (d) HEK293 cells were transfected with expression constructs for FL-TRAIL (FL), secreted TRAIL (sT) or an empty plasmid (ctrl). After 48 h the supernatants were filtered through a 0.45 μm filter and the resulting filtrates used for a TRAIL ELISA. Values represent mean ± SE.