Figure 1. The requirement of Vps33B for phagosome maturation is conserved.

A) Map of Vps33B flanked by an alternative Fur1 noncoding exon and the neighboring CG4553 gene. The Vps33B^B5.20 deletion allele removes the alternative Fur1 noncoding exon, the Vps33B promoter and codons for the first 196 amino acids of Vps33B. A transgene containing P[acman]Ch322-65H17 rescued all phenotypes of Vps33B^B5.20 discussed here, but not a linked “blemished wing” phenotype.

B) Micrographs of primary hemocytes that phagocytosed GFP-labeled E. coli for 20 min and were chased for 45 min.

C) Quantification of phagocytosed E. coli detectable after 10-min or 45-min chases. Genotypes in B, C: were OreR, w^1118;+/+;Vps33B^5.20, and w¹¹¹⁸; BAC^{CH322-65H17/+}; Vps33B^5.20. (***: p<0.001, One way ANOVA)

D) Immuno blots of macrophages expressing none, scrambled control or Vps33B shRNA developed with Vps33B antibodies. Alpha-tubulin served as loading control.

E) Quantification of Vps33B protein from western blots as shown in D (n = 3).

F,G) Micrographs of macrophages expressing scrambled control (F) or Vps33B shRNA (G) allowed to phagocytose GFP-labeled E. coli for 20 min followed by a 45-min chase.

H) Quantification of phagocytosed E. coli detectable after a 45-min chase as shown in F and G (***: p<0.001, Two-tailed t-test).

I) Flow cytometry analysis of macrophages expressing control or Vps33B shRNA allowed to phagocytose GFP-labeled E. coli for 20 min followed by a chase of the indicated times. [All data here are representative of at least 3–5 independent experiments.

Please also see Figure S1.