tPA slows the release of co-packaged PAI-pHl and NPY-pHl. Bovine chromaffin cells were cotransfected to express cargo proteins tagged with pHl in tandem with tPA, PAI, or pCDNA3 vector control. Secretion was stimulated with 56 mM potassium buffer and observed by TIRF microscopy at a rate of 36 Hz. pHl fluorescence intensity was analyzed with a custom program as described in Fig. 5. (A and B) tPA slows PAI-pHl secretion. (C and D) tPA slows NPY-pHl secretion. (E and F) PAI does not slow the secretion of tPA-pHl. Each dot represents one fusion event, and medians are indicated by lines. Cumulative histograms plot data from upper panels. A Kolmogorov–Smirnoff test was performed for A and C. One-way ANOVA was performed in E with a post hoc Kruskal–Wallis test.