Figure 5.

Reduction of both rab6A and A' by RNA interference (RNAi) effects Golgi structure and transport. (A) Western blot demonstration of reduced total rab6 protein levels by two different siRNA sequences (labeled A/A'-siRNA1 and 2) or reduced rab6A or A' protein upon addition of the respective isoform-specific duplex (labeled A-siRNA and A'-siRNA). Whole cell extracts were prepared 92 h after repeated transfection with the appropriate siRNA and analyzed by Western blotting with anti-rab6 mAb. Levels of rab6 in cells treated with control-siRNA (Ctrl-siRNA), and levels of α-tubulin were not affected. (B) Suppression of individual rab6 isoforms by isoform-specific siRNA sequences. Cells treated with siRNA for 22 h were transiently transfected with plasmids encoding GFP-rab6A or GFP-rab6A' and prepared for immunoblot 28 h postplasmid transfection. The anti-GFP immunoblot shows the efficiency and specificity of each siRNA under these conditions. Thus, each GFP-rab6 isoform is not expressed when targeted by its siRNA and is expressed when incubated with siRNA against the other isoform. siRNA targeting both rab6A and A' inhibited expression from GFP-rab6A and GFP- rab6A' plasmids as expected. GFP-rab6 expression in cells treated with an unrelated control siRNA and α-tubulin were not affected. (C-J) Immunofluorescence images of cells expressing the Golgi marker GalNAc-T2^GFP after double treatment with the corresponding siRNAs for 92 h and immunostaining with rabbit anti-rab6 antibody. These illustrations were imaged with the same camera exposure settings, allowing a direct comparison of rab6 levels after RNAi treatment (C-F). The minor effect of reduced levels of rab6A (D and H) or A' (E and I) on Golgi structure. Reduction of rab6A and A' combined (F and J) affects overall Golgi morphology compared with control-treated cells (C and G). Arrowheads indicate the cells in which there was a major reduction in Golgi size. (K-N) Relocalization of GalNAc-T2^GFP to the ER after the introduction of Sar1p (GTP) is delayed in rab6 knockdown cells. Stably transfected GalNAc-T2^GFP cells treated with Ctrl-siRNA (K and L) or A/A'-siRNA1 (M and N) for 92 h were injected with DNA encoding dominant negative Sar1p (asterisks) and incubated for 4 h. Cells were then processed for double labeling with antibodies directed against rab6 (our unpublished data) and Sar1p (L and N) and observed by microscopy. Cells were analyzed for the amount of GalNAc-T2^GFP redistribution (K and M). Bar, 10 μm.