Multiple oxidoreductases secreted by unstimulated endothelial cells. (A) HUVECs grown to confluence on a 96-well plate, washed thoroughly, and then incubated with 30 μL/well of HBS buffer for 30 minutes at 37°C. HBS supernatant was then removed, and cells were lysed with a volume of lysis buffer equal to supernatant. Lysate neat (20 μL) or 20× concentrated HBS supernatant was then transferred to PVDF and probed for the relevant oxidoreductase. (B) HBS was incubated with HUVECs as above for 5 and 30 minutes, and neat supernatant was probed for PDI. Amount of PDI detected is expressed as arbitrary units. (C) HUVECs and EAhy926 cells were grown to confluence in parallel within the same 96-well plate, washed, and incubated with HBS buffer (30 μL/well) for 30 minutes. The buffer supernatant was then removed, and equal amounts of supernatant were transferred to PVDF membrane and probed with an anti–TRX-R antibody. A cell viability assay confirmed equivalent amounts of viable cells for HUVECs and EAhy926 cells. TRX-R was only detectable in HUVEC supernatant after concentration 20×, as shown in panel A. All blots are representative of 3 independent experiments.