Figure 7.

Dominant-negative forms of Rab5 or Rab7 alter the association of retromer with endosomes. (A–I) HeLa cells were transfected with plasmids encoding GFP-Rab4a–S22N (A–C), GFP-Rab5a–S34N (D–F), GFP-Rab7–T22N (G–I), or GFP-Rab7–Q67L. At 8 h after transfection, the cellular distribution of Vps26 (B, E, and H) and SNX1 (C, F, and I) was analyzed by indirect immunofluorescent staining using rabbit polyclonal antibody to Vps26 and mouse monoclonal antibodies to SNX1 followed by Alexa Fluor 594–conjugated donkey anti–rabbit IgG and Alexa Fluor 647–conjugated donkey anti–mouse IgG. (J–L) Cells were treated with 200 nM wortmannin in DMSO for 30 min at 37°C and the distribution of Vps26 and SNX1 was analyzed by indirect immunofluorescent staining using the respective primary antibodies and fluorescently labelled secondar)y antibodies. Images were captured by epifluorescence microscopy. Arrows point to cells expressing the GFP-Rabs. Bar, 10 μm.