AP-3 RNAi diverts SgII to constitutive secretory vesicles budding from the TGN. (A and B) PC12 cells were transfected twice with 50 nM AP-3 or control siRNA, labeled for 5 min with 0.5–1 mCi/ml ³⁵SO₄, and either harvested directly (pulse [p]) or incubated at 37°C for 4 h (chase [c]) in complete medium containing 1.6 mM nonradioactive Na₂SO₄. (A) Separation of duplicate cell extracts by electrophoresis followed by autoradiography shows that after 4 h, control cells store almost all of the labeled SgII. In contrast, AP-3 RNAi dramatically reduces the storage of SgII with no effect on the constitutively secreted HSPG. Error bars indicate the percent decline in cellular content of labeled protein after the chase relative to just after the pulse. *, P < 0.02 relative to control by two-tailed Student’s t test (n = 3). (B) Aliquots of media from the times indicated confirm the constitutive secretion of HSPG unaffected by AP-3 RNAi and the constitutive release of SgII produced by AP-3 RNAi. (C) PC12 were transfected and labeled with ³⁵SO₄ as described in A, incubated at 37°C for 15 min after the pulse, and a postnuclear supernatant separated by velocity sedimentation through a 0.3–1.2 M continuous sucrose gradient, with the TGN-derived vesicles in fractions 1–5 separated further by equilibrium sedimentation on a 0.5–2 M continuous sucrose gradient. (left) 500 µl fractions were collected from the top of the gradient, and 50 µl aliquots were separated by electrophoresis followed by fluorography. (right) Only fractions with significant amounts of radioactivity are shown, and the labeled HSPG and SgII in each fraction are expressed as a percentage of the total labeled protein on the gradient. The amount of SgII in the HSPG peak increases with AP-3 RNAi, and the bar graph indicates the area under the HSPG peak as a percentage of the area under the entire SgII curve (inset). *, P < 0.05 relative to control by two-tailed Student’s t test. The data indicate means of three independent transfections, and error bars indicate SEM.