AP-3 RNAi increases VMAT2 surface expression and impairs regulated release of SgII from PC12 cells. (A and B) Western analysis of extracts from PC12 cells transiently transfected with 50 nM AP-3 siRNA show an ∼80% reduction in AP-3δ subunit σ3A relative to cells transfected with control siRNA (A). PC12 cells cotransfected with wt or EE/AA GFP-/HA-VMAT2 with or without AP-3δ siRNA were subjected to flow cytometry as described in Fig. 1 B. Kolmogorov-Smirnov analysis of the cumulative frequency distributions for wt VMAT2 + control siRNA (gray), wt VMAT2 + AP-3δ siRNA (red), and EE/AA VMAT2 + control siRNA (black) indicates a significant change in the AP-3 siRNA distribution relative to wt (P < 10⁻¹⁴). (C and D) PC12 cells were transiently transfected with AP-3δ or control siRNA, washed, and incubated for 30 min in Tyrode’s solution containing 2.5 mM (basal) or 90 mM (stimulated) K⁺. Cellular and secreted SgII were measured by quantitative fluorescent immunoblotting (C), with the secreted SgII normalized to basal secretion in the control (D). AP-3δ RNAi greatly reduces the depolarization-induced secretion of SgII. *, P < 0.05 relative to stimulated secretion from control by two-tailed Student’s t test (n = 4). (E) AP-3δ RNAi reduces the cellular content of SgII relative to actin. *, P < 0.005 relative to control by two-tailed Student’s t test (n = 4). (F) The adrenal glands of mocha mice lacking AP-3 show a dramatic reduction in the content of SgII and chromogranin A (CgA) relative to the adrenals of control littermates. *, P < 0.05; **, P < 0.005 (relative to wt by two-tailed Student’s t test; n = 3). The data show mean values, and error bars indicate SEM.