BFA- and CHX-insensitive stimulated secretion and storage efficiency of tPA and cytokines in HUVECs. (A) Top panels: Histamine-stimulated secretion of tPA (i), IL-8 (ii), IL-6 (iii), MCP-1 (iv), and GRO-α (v) in the absence (BFA vehicle, 1 hour) or after BFA treatment (1 hour) as indicated. Cells were pretreated for 24 hours with either 3mM Na-butyrate (i) or 1 ng/mL rhIL-1β (ii-v) before experiments. Bottom panels: Histamine- or ionomycin-stimulated secretion of tPA and the cytokines in control (CHX vehicle for 24 hours) or CHX-treated (24 hours) cells as indicated. Na-butyrate or rhIL-1β was included in the media during CHX treatment. Data shown in each case are an individual experiment, carried out in triplicate, and are representative of 3 or 4 replicate experiments. *P < .05. **P < .001. (B) Storage efficiency for VWF, tPA, and lumEGFP determined by pulse-chase methodologies and for lumEGFP, IL-8, IL-6, MCP-1, and GRO-α determined by specific ELISA methodologies as indicated. Pulse-chase data in each case are the mean of 2 experiments each carried out in triplicate. Error bars represent the range of the duplicate values. ELISA data are the mean of 3 or 4 separate experiments carried out either in triplicate (LumEGFP) or in duplicate (the cytokines). (C) lumEGFP fluorescence (left panel) and VWF immunoreactivity (right panel) in a cell 48 hours after Nucleofection with lumEGFP. Arrowheads indicate lumEGFP-positive WPBs.