S2 cells express an RSP. (A and B) S2 cells were transiently transfected with wt (black) or DE/AA (red) GFP- and HA-tagged dVMAT, incubated for 2 h at room temperature with external HA antibody conjugated to Alexa Fluor 647, washed, and the fluorescence of individual cells was determined by flow cytometry (A). (inset) The bar graph displays the mean ratio of surface/total fluorescence (n > 1,800 cells). Kolmogorov-Smirnov analysis of the cumulative frequency distributions binned by surface/total dVMAT ratios (B) indicates a significant change in the DE/AA distribution relative to wt (***, P < 10⁻¹⁴). (C–F) S2 cells were transiently transfected with ANF-GFP (C–E) or the signal sequence of ANF fused directly to GFP (ss-GFP; C and D), washed, incubated for 1 h, and the cellular and secreted GFP fluorescence were measured using a plate reader. (C) Normalized to intracellular fluorescence, the media of cells expressing ANF-GFP shows less fluorescence than cells expressing ss-GFP. (D) Treatment with 100 µg/ml LPS induces secretion of ANF-GFP but not ss-GFP. (E and F) Pretreatment with BAPTA-AM in calcium-free buffer (E) or knockdown of Drosophila calcium-dependent activator protein for secretion (dCAPS) with dsRNA (F) block the secretion evoked by LPS. *, P < 0.05 by two-tailed Student’s t test. The data represent mean values from three to four independent experiments, and error bars indicate SEM.