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. 2003 Oct 27;163(2):339–350. doi: 10.1083/jcb.200307046

Figure 1.

Figure 1.

Activated Rab8 (but not dominant-negative Rab8) mislocalizes VSV-G to the apical surface. (A) Fully polarized MDCK cells were microinjected with cDNAs of ts045 VSV-G GFP and T7-tagged Rab8Q67L (dominant active) or Rab8T22N (dominant negative) at 200 ng/μl, incubated at the nonpermissive temperature of 40°C to accumulate VSV-G in the ER for 2 h, and chased for 2 h at the permissive temperature of 31°C in the presence of cycloheximide. The cells were fixed, permeabilized, and processed for IF. Indirect IF of an endogenous basolateral protein using the anti-gp58 antibody (red, second row), followed by Alexa® 568 secondary antibody. The cells were analyzed by confocal microscopy and a representative x-z section is shown. (B) Polarized MDCK cells were microinjected with the cDNAs of apical ts045 VSV-G–G3 with T7-Rab8Q67L or T7-Rab8T22N under the same pulse-chase conditions as above. Cells were processed for IF as above. Rab8 expression (red, second row) was monitored using a mouse anti-T7 tag antibody followed by anti–mouse Alexa® 568 secondary antibody. (C) ts045 VSV-G–GFP was microinjected with T7-Rab8Q67L under the same pulse-chase conditions as in A, but the chase is followed by fixation without permeabilization. The cells are immunolabeled using an antibody, TK-G, against the ectodomain of VSV-G followed by Alexa® 568 secondary antibody. (D) Confluent MDCK cells were infected overnight at 40°C with adenoviruses encoding ts045 VSV-G–GFP and T7-Rab8Q67L. After a 2-h chase at 31°C in the presence of cycloheximide, the cells were trypsinized, fixed without permeabilization, and immunolabeled using the TK-G antibody followed by an anti–mouse phytoerythrin secondary antibody. FACS® analysis shows that the efficiency of surface expression of VSV-G is unchanged in the presence of Rab8Q67L.