Figure 5.

RNAi-mediated depletion of Rab7 causes Vps26 dissociation from endosomes. (A) HeLa cells were treated twice at 24-h intervals with a control, inactive siRNA, or siRNAs to Rab7, Rab4 (targeting both the a and b isoforms), or Vps26. Cell extracts were analyzed by SDS-PAGE and immunoblotting with antibodies to Rab7, Rab4, Vps26, or actin (loading control) as indicated in the figure. (B–Q) HeLa cells treated as in A with control (B, C, F, G, J, K, N, and O) or Rab7 siRNA (D, E, H, I, L, M, P, and Q) were immunostained for Vps26 (B, D, F, H, J, L, N, and P) and AP-3 (C and E), AP-1 (G and I), SNX1 (K and M), and SNX2 (O and Q) using rabbit polyclonal antibody to Vps26 and mouse monoclonal antibodies to AP-3–δ, AP-1–γ1, SNX1, or SNX2 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG and Alexa Fluor 488–conjugated donkey anti–mouse IgG. Images were captured by epifluorescence microscopy. Bar, 10 μm.