Overexpression of syt IV has similar effects on secretion from the cells as upregulation of syt IV. (A) Confocal images of cells are shown stained with fluorescently labeled antibodies to syt IV (red), EGFP (green), and nuclei (blue). The merge of all three images is shown on the right. The top group of images shows endogenous staining for syt IV in control cells that have not been transfected with the plasmids that express syt IV and EGFP. The lower group of images shows cells cotransfected with syt IV/EGFP plasmids in a 10:1 ratio. The images are representative of 3 independent experiments (scale bar = 10 μm). (B) Amperometry recordings are shown in a representative control cell transfected with the plasmid that expressed syt IV (pSyt IV) (C) and in an shRNA-syt I knockdown cell transfected with the pSyt IV (B). The arrows depict where the continuously perfused solution was changed from HBSS to 50 mM K+ stimulating solution. (D) The average number of events is plotted for control cells (n = 16) and two independent stable syt I knockdown cell lines (syt Ia, n = 16, and syt Ib, n = 20) compared to cells cotransfected with human syt IV/EGFP (control, n = 13 cells; syt Ia, n = 17 cells; and syt Ib, n = 17 cells; *p < 0.05 and **p < 0.01). (E) Basal and stimulated norepinephrine (NE) release were analyzed from 9-21 independent experiments. NE release is expressed as total NE (ng) per 50 μl sample (**p < 0.01).