Figure 5. AP-1 knockdown potentiates the effect of AP-3 knockdown on regulated secretion.

(A) PC12 cells were transfected with pooled siRNA directed against AP-1 γ or pooled nontargeting siRNA as control. Knockdown was assessed by quantitative fluorescent immunoblotting for AP-1 γ with actin as loading control. (B) PC12 cells were transfected with control, AP-1 γ, AP-3 δ or both siRNAs, washed, and incubated for 30 min in Tyrode's solution containing 2.5 mM (basal) or 50 mM (stimulated) K⁺. Secreted SgII was measured by quantitative fluorescent immunoblotting and normalized to basal secretion from cells treated with control siRNA. AP-1 γ RNAi alone reduces the depolarization-induced secretion of SgII very similar to AP-3 δ RNAi, and combined AP-1 γ/AP-3 δ RNAi potentiates the decrease in regulated secretion. (C) Cells were lysed and intracellular SgII measured as above, normalizing to actin. AP-1 γ and AP-3 δ RNAi both reduce cellular SgII, but the combined knockdown does not show an additional effect. (D) Secreted SgII was normalized to cellular SgII levels for each RNAi condition, and expressed relative to basal secretion from cells treated with control siRNA. Normalizing to cellular SgII shows a significant increase in basal secretion of SgII with AP-1 γ RNAi whereas AP-3 δ RNAi significantly reduces depolarization-induced secretion. Combined AP-1 γ/AP-3 δ RNAi potentiates the reduction in stimulated release. *p<0.05; **p<0.01; ***p<0.001 (Newman-Keuls post-hoc test; n = 4 transfections). The data show mean values, and error bars indicate s.e.m.