Western blot analysis of soluble forms of recombinant tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), enzymatic activity deficient form of fused protein (sTRAIL:mFeSOD) and sTRAIL:iron superoxide dismutase (FeSOD) expression and sTRAIL:FeSOD-induced cell death. (A) After isopropyl-β-D-thiogalactoside induction, the expressed sTRAIL, sTRAIL:mFeSOD or sTRAIL:FeSOD was subjected to Western blot analysis with anti-TRAIL antibody. Lanes 1 to 5 are sTRAIL control (Peprotech, Rocky Hill, New Jersey, USA) and extracts of BL-21 transformed with pET28, pET28-sTRAIL, pET28-sTRAIL:mFeSOD and pET28-sTRAIL:FeSOD, respectively. (B) Samples were prepared and electrophoresed using native polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions followed by electrotransfer and immunoblotting with TRAIL antibody. Lanes 1 to 3 are sTRAIL control (purchased from Peprotech), sTRAIL:FeSOD (before renaturation) and sTRAIL:FeSOD (renatured), respectively. (C) Six-well plates were seeded with LO2 cells and allowed to adhere before replacement with RPMI 1640 medium with hyperosmotic sucrose (0.25 M) containing sTRAIL (500 ng/ml), sTRAIL:FeSOD (1,000 ng/ml) or sTRAIL:mFeSOD (1,000 ng/ml). After treatment for 8 hours, cell death was quantified by flow cytometry. (D) through (G) Cells were grown in six-well plates to 60% confluence, and the indicated proteins were added at the indicated concentrations. After treatment for 8 hours, cells were stained with fluorescein isothiocyanate anti-annexin V antibody and propidium iodide and then examined using flow cytometric analysis. For each sample, 10,000 events were acquired. Results are expressed as the mean fluorescence intensity. Each bar represents the mean ± SE obtained from three independent experiments.