Depletion of Rab7 blocks the transport of CI-MPR from endosomes to the TGN. HeLa cells were treated twice at 24-h intervals with an inactive siRNA (control; A, C, E, and G) or siRNA to Rab7 (B, D, F, and H–N). At 48 h after treatment, the steady-state distribution of CI-MPR (A–D) was assessed by indirect immunofluorescent staining of fixed cells using rabbit polyclonal antibody to cytosolic tail of CI-MPR followed by Alexa Fluor 488–conjugated donkey anti–rabbit IgG. C and D correspond to high magnification views of CI-MPR–positive structures from control or Rab7-depleted cells, respectively. Live control cells (E and G) or Rab7-depleted cells (F and H–N) were incubated with an antibody to the luminal domain of CI-MPR for 2 h at 37°C. Cells were washed, fixed, permeabilized, and stained with Alexa Fluor 488–conjugated donkey anti–mouse IgG to detect internalized antibody to CI-MPR (E–H, J, and M). G and H show high magnification views from control (E) or Rab7-depleted cells (F), respectively. Cells in I–N were additionally stained with rabbit polyclonal antibody to TfR (I–K) or giantin (L–N) followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG. Images in A–H were captured using an epifluorescence microscope, and images in I–N were captured with a confocal microscope. (K and N) For merged images, yellow indicates colocalization. Arrows in I–K indicate examples of foci where proteins colocalize. (O) Extracts of HeLa cells treated with siRNAs to the proteins indicated on top were analyzed by 4–20% acrylamide gradient SDS-PAGE and immunoblotting (IB) with antibodies to the proteins indicated on the right. Equal amounts of total protein were loaded. Bars: (A, B, E, and F) 15 μm; (C, D, G, and H) 1.5 μm; (I–N) 10 μm.