(a–c) Colocalization of GFP-Rab9 and Serp in the tracheal cells at late stages of embryogenesis (stages 15 and 16, white arrowheads in a,b). Insets in a and b show GFP-Rab9 endosomes containing Serp. The colocalization rate increased as development proceeded (c). (d) Tracheal cells expressing RFP-Rab9 and Serp-chitin-binding domain-GFP (Serp-CBD-GFP) driven by btl-GAL4, showing colocalization of the two markers (arrowheads). Inset shows enlargement of endosome. (e) Western blotting of whole-cell lysates from 20 embryos probed with anti-Serp antibody. 1, btl-GAL4>GFP-Rab9; 2, Df(2L) Exel8041/+; 3, Df(2L)Exel8041 homozygote; 4, rab956/+; 5, rab956 homozygote; 6, rab9199/+; and 7, rab9199 homozygote. (f–m) Control heterozygotes (f,h) and rab956/rab9199 (g,i) embryos at stage 14 and 16 were immunostained with Serp and CBP. White arrowheads in f–i show the expression level of Serp in tracheal tubes in rab9-mutant embryos was unaltered at stage 14 (j,l) and apparently decreased at stage 16 (k,m) compared with control embryos. Blue arrowheads (k,m,n–q) show the significant reduction of Serp protein in the rab9-mutant tracheal lumen, whereas the chitin expression showed no difference between the control and rab9-mutant embryo (yellow arrowheads in k,m). (n,p) Stage-16 rab9-mutant heterozygous (n) and homozygous (p) embryos immunostained for Serp and Uninflatible (Uif). White arrowheads in p show Serp colocalized with apical membrane marker Uif. (o,q) Stage-16 rab9-mutant heterozygous (o) and homozygous (q) embryos immunostained for Serp and 2A12 antigen. Blue arrowheads show Serp significantly decreased in the lumen, but 2A12 does not show detectable difference between rab9 mutant and the control. Scale bar, 10 μm.