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. 2005 Oct;16(10):4495–4508. doi: 10.1091/mbc.E04-12-1042

Figure 5.

Figure 5.

Decreased expression of Arf1+Arf4 and Arf1+Arf3 altered traffic of ts045-VSVG-GFP. HeLa cells were cotransfected with pSUPER-based plasmids on day 0. After 2 d, cells were transfected with the plasmid directing expression of ts045-VSVG-GFP. Cells were then incubated at the restrictive temperature (40°C) for 16 h before switching to the permissive temperate (32°C) for 90 min before fixing, before collection of confocal images. (A) Equal amounts of pSUPER control or the two plasmids directing knockdown of pairs of Arfs, as indicated in each panel, were used for transfections. Nonpermeabilized cells were incubated with ConA-Alexa594 to label the cell surface. Colocalization of ts045-VSVG-GFP (green) and ConA (red) was visualized as yellow in the merged images. In Arf1+Arf4 knockdown cells, ts045-VSVG-GFP displayed an ER-like appearance, did not localize to the cell surface and did not colocalize with ConA. In Arf1+Arf3 knockdown cells, ts045-VSVG-GFP localized to enlarged puncta. Although ts045-VSVG-GFP traveled to the cell surface in these cells (arrows), the amount of staining at the cell surface seemed diminished. Bar, 10 μm. (B) Percentage of cells in which ts045-VSVG-GFP showed a reticular/ER distribution throughout the entire cell (i.e., seemed similar to the distribution of the ER marker calnexin) was quantified (pSUPER, n = 100; Arf1+Arf3, n = 100; Arf1+Arf4, n = 150; Arf1+Arf5, n = 100; Arf3+Arf4, n = 100; Arf3+Arf5, n = 100; Arf4+Arf5, n = 150). The percentage of cells displaying fluorescence at the plasma membrane also was quantified (pSUPER, n = 150; Arf1+Arf3, n = 100; Arf1+Arf4, n = 150; Arf1+Arf5, n = 100; Arf3+Arf4, n = 100; Arf3+Arf5, n = 100; and Arf4+Arf5, n = 100). Cells were scored a 0 if they showed no cell surface localization and a 1 if they showed any localization to the plasma membrane regardless of the intensity. Therefore, these cell counts are a conservative estimate of whether ts045-VSVG-GFP travels to the plasma membrane under these conditions but do not reflect the amount of ts045-VSVG-GFP at the cell surface.