a, b) S2 cells expressing GFP-Rab9 and Vps35-mRFP. Yellow and white arrowheads show the colocalization of Vps35-mRFP with GFP-Rab9-enriched membrane domains of small endosomes and vacuolar endosome, respectively. ( c) Fluorescent intensity of GFP-Rab9 (green) and Vps35-mRFP (magenta) in an endosomal membrane. ( d) Budding of GFP-Rab9 endosomes in S2 cells. White arrowheads indicate small vesicle formation, budding and release from the endosome ( Supplementary Movie 1). ( e) Tracheal cells co-expressing GFP-Rab9 and myc-Vps35. White arrowheads in the insets indicate myc-Vps35 puncta localized on the Rab9 endosomal membrane. ( f) Colocalization of Serp-GFP with RFP-Rab9 in S2 cells. White arrowheads indicate that the Serp-GFP colocalizes with RFP-Rab9 in the endosomal membrane. Blue arrowheads indicate the colocalization of Serp-GFP puncta with small RFP-Rab9 vesicles in the cytoplasm. ( g, h) S2 cells were co-transfected with GFP-Rab7 and Vps35-mRFP, then treated with control ( g) and Rab9 ( h) dsRNA. ( i, j) S2 cells were co-transfected with GFP-Rab7 and RFP-Rab9, then treated with control ( i) and Vps35 ( j) dsRNA. Yellow arrowheads in ( i) showed the tubule structures extending from endosomes. ( k) Reverse transcription PCR of Rab9 and Vps35 fragment from the control and dsRNA-treated cells. rp49 was amplified for the internal control. Scale bar, 10 μm for a, b and e– j and 2 μm for d.