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Published in final edited form as: Neurobiol Aging. 2012 Jun 5;33(9):2231.e15–2231.e30. doi: 10.1016/j.neurobiolaging.2012.04.020

Loss of Snx3 and Rab7A both affect retromer-association but through different mechanisms. A. HeLa cells stably expressing GFP-Snx3 were treated with siRNA to abolish Snx3 or Rab7A expression. Loss of Snx3 or Rab7A result in a similar reduction in VPS26 labeling but loss of Rab7 appears to increase the intensity of the GFP-Snx3 labeling. Bar = 20µm. B. HeLa cells stably expressing GFP-Rab7A were treated as in A. In this experiment, loss of Snx3 expression reduces the punctate labeling of the GFP-Rab7A and also results in a loss of VPS26 labeling. The GFP-Rab7A construct is of murine origin and is therefore resistant to the siRNA targeting human Rab7A and hence VPS26 labeling is similar to control cells. Bar = 20µm. C. Lysates from cells shown in A and B were subjected to SDS-PAGE and analyzed by western blotting. The Rab7A knockdown increases the GFP-Snx3 levels consistent with the increased intensity of GFP-Snx3 signal observed in A. D. Cells stably expressing various GFP-tagged endosomal proteins were treated with siRNA to abolish Snx3 expression. VPS26-positive endosomes were then quantified using the Cellomics ArrayScan microscope. In all cases, loss of Snx3 expression results in a marked reduction in the number of VPS26-positive endosomes indicating that increased expression of Rab7A or the constitutively active Rab7A Q67L mutant is unable to rescue the loss of Snx3. For comparison, the number of VPS26-positive endosomes determined for control and Snx3 KD cells in Figure 2C is shown by the dotted lines. E. Cells stably expressing GFP-Snx3 or GFP-Rab7A were treated with siRNA to KD Snx3 or Rab7A expression. Lysates were immunoprecipitated using anti-GFP and following SDS-PAGE, VPS35 and VPS26 were detected by western blotting. The KD of Snx3 abolishes the association of retromer with GFP-Rab7A, loss of Rab7A expression reduces but does not completely abolish the interaction between GFP-Snx3 and retromer.

Loss of Snx3 and Rab7A both affect retromer-association but through different mechanisms. A. HeLa cells stably expressing GFP-Snx3 were treated with siRNA to abolish Snx3 or Rab7A expression. Loss of Snx3 or Rab7A result in a similar reduction in VPS26 labeling but loss of Rab7 appears to increase the intensity of the GFP-Snx3 labeling. Bar = 20µm. B. HeLa cells stably expressing GFP-Rab7A were treated as in A. In this experiment, loss of Snx3 expression reduces the punctate labeling of the GFP-Rab7A and also results in a loss of VPS26 labeling. The GFP-Rab7A construct is of murine origin and is therefore resistant to the siRNA targeting human Rab7A and hence VPS26 labeling is similar to control cells. Bar = 20µm. C. Lysates from cells shown in A and B were subjected to SDS-PAGE and analyzed by western blotting. The Rab7A knockdown increases the GFP-Snx3 levels consistent with the increased intensity of GFP-Snx3 signal observed in A. D. Cells stably expressing various GFP-tagged endosomal proteins were treated with siRNA to abolish Snx3 expression. VPS26-positive endosomes were then quantified using the Cellomics ArrayScan microscope. In all cases, loss of Snx3 expression results in a marked reduction in the number of VPS26-positive endosomes indicating that increased expression of Rab7A or the constitutively active Rab7A Q67L mutant is unable to rescue the loss of Snx3. For comparison, the number of VPS26-positive endosomes determined for control and Snx3 KD cells in Figure 2C is shown by the dotted lines. E. Cells stably expressing GFP-Snx3 or GFP-Rab7A were treated with siRNA to KD Snx3 or Rab7A expression. Lysates were immunoprecipitated using anti-GFP and following SDS-PAGE, VPS35 and VPS26 were detected by western blotting. The KD of Snx3 abolishes the association of retromer with GFP-Rab7A, loss of Rab7A expression reduces but does not completely abolish the interaction between GFP-Snx3 and retromer.