Characterization of CD63 and Tsp29Fa in salivary gland cells. (A, B, and E–G) Spinning-disc confocal images of L3 salivary gland cells. (A and B) Cells from early (A) and late (B) L3 expressing CD63-GFP and Tsp29Fa-Flag-HA driven by one copy of AB1-GAL4, fixed and stained for GFP (green) and HA (red). (C and D) Colocalization analysis of CD63-GFP and Tsp29Fa-Flag-HA from early (C) and late (D) L3 salivary gland cells. Error bars indicate standard deviation. (C) Pearson’s = 0.43 ± 0.13, M(CD63) = 0.84 ± 0.10, M(Tsp29Fa) = 0.36 ± 0.10. (D) Pearson’s = 0.22 ± 0.041, M(CD63) = 0.51 ± 0.084, M(Tsp29Fa) = 0.47 ± 0.14. Analysis was performed on a total of five images from two independent experiments. (E) Live cells expressing Sgs3-GFP (grayscale) in control cell (left) or cell with one copy of Tsp29-Flag-HA and one copy of CD63-GFP driven by two copies of AB1-Gal4 driver (right). (F) Live salivary gland cells expressing CD8-GFP (left) or CD63-GFP and CD8-RFP (right) with AB1-Gal4 driver. Magenta arrows indicate apical membranes in confocal and corresponding merged fluorescence and bright-field images. (G) Salivary gland cells without (left) or with (right) one copy of Tsp29Fa-Flag-HA driven by AB1-Gal4. Cells were fixed and stained for Hrs (green, EE), Lva (blue, cis-Golgi), and HA (red, Tsp29Fa-Flag-HA). Dashed boxes mark regions magnified 2.5-fold in insets. Magenta arrows show EE adjacent to cup-shaped cis-Golgi structures.