**AP-3 RNAi affects the properties of LDCVs.** (A and B) PC12 cells were transfected twice with 50 nM AP-3δ or control siRNA, and the postnuclear supernatant (input) obtained 2–3 d after the second transfection was separated by equilibrium sedimentation through 0.6–1.6 M sucrose. Fractions were collected from the top of the gradient and assayed for synaptophysin (syp) and SgII by quantitative fluorescent immunoblotting, with each fraction expressed as the percentage of total gradient immunoreactivity, and the area under the LDCV peak (black lines) expressed as a percentage of the area under the entire curve (inset). (A) AP-3 RNAi greatly reduces the LDCV peak and shifts the SgII immunoreactivity toward lighter fractions without affecting the synaptic vesicle protein synaptophysin. *, P < 0.05 relative to control by two-tailed Student’s *t* test (*n* = 3 transfections). (B) PC12 cells were cotransfected with ANF-GFP and either AP-3 or control siRNA, and the postnuclear supernatant was sedimented as in A. In this case, however, ∼80 fractions were collected from the top of the gradient directly into a 96-well plate, and the fluorescence of ANF-GFP was measured directly using a plate reader. The graph indicates ANF-GFP fluorescence for each fraction expressed as a percentage of total gradient fluorescence. (right) The bar graph shows the area under the curve for the LDCV peak (black line), expressed as a percentage of total area. *, P < 0.01 relative to control by two-tailed Student’s *t* test (*n* = 3 transfections). (C and D) PC12 cells were transfected twice with either control or AP-3 siRNA and processed for electron microscopy 2 d after the second transfection. (C) Low magnification electron micrographs show a large reduction in the number of LDCVs (arrowheads) of cells transfected with AP-3 siRNA (right) relative to controls (left). Bar graphs indicate the number of LDCVs per cell in the section (left) and LDCV density (right). *, P < 0.0005; **, P < 0.000001 (*n* = 20 cells/condition). (D) Higher magnification electron micrographs show that AP-3 RNAi increases the size of LDCVs. Bar graphs indicate the corrected diameters (left) and areas (right) of both the entire LDCV and the electron-dense core (Parsons et al., 1995). (bottom) The LDCV area is presented as a frequency histogram. *, P < 0.01 (*n* = 205–221 LDCVs/condition). Bars, 200 nm.

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