Fig. 10. The VPS9 domain and ankyrin repeats are required for the subcellular localization and activity of Varp in HeLa cells. (A) Schematic diagram of the Varp protein and its mutants used in this experiment. (B) Cell lysates from cells expressing EGFP-fused Varp proteins were analyzed by western blotting with anti-GFP and anti-Varp antibodies. (C) Distribution of Varp and its mutants. HeLa cells ectopically expressing EGFP-fused proteins were fixed and labeled with an anti-EEA1 antibody (red); (c,f,i,l,o) show the co-staining of Varp or mutants with the early endosomal marker EEA1. The effect of the full-length or Varp mutants on the size of early endosomes was also analyzed (b,e,h,k,n). Bars, 10 µm. Arrows indicate the colocalization of Varp and EEA1 on the enlarged early endosomes; ^*, giant late endosome. (D) Colocalization of Varp/VPS and late endosomal markers. (a-c) HeLa cells transfected with Myc-tagged Varp/VPS and EGFP-tagged Rab5 were stained with an anti-Myc antibody (red). (d-f) HeLa cells transfected with Myc-tagged Varp/VPS and EGFP-tagged Rab7 were stained with anti-Myc antibody (red). (g-i) HeLa cells expressing EGFP-tagged Varp/VPS were stained with an anti-LAMP3 antibody (red). Bars, 10 µm.