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Fig. 5. Trafficking of transmembrane proteins is inhibited in GBF1-depleted cells. (A-C) HeLa cells were transfected with scrambled siRNA (scr) or siRNA against GBF1 for 48 hours. Cells were then transfected with ts045VSV-G-GFP and cultured for an additional 
12 hours at 42°C. Cells were shifted to the permissive temperature of 32°C for 0, 1 or 2 hours. At each time point, cells were processed for immunofluorescence with antibodies against GM130. VSV-G is visualized by green fluorescence. GBF1-depleted cells (indicated by asterisks) are visualized by tubulation of GM130. In GBF1-depleted cells, trafficking of VSV-G is inhibited. (D) The kinetics of VSV-G processing to the Endo-Hf-resistant Rt form were quantitated from fluorographs as described in Materials and Methods and are presented as the percentage of total VSV-G present at each time point. A significant reduction in VSV-G trafficking in GBF1-depleted cells is observed. (E-G) HeLa cells were untreated (E), transfected with scrambled siRNA (F) or siRNA against GBF1 (G) for 72 hours and stained with the indicated antibodies. In control cells, ESL-1 colocalizes with fibronectin in thin connections between cells. Similar surface localization is seen in cells transfected with scrambled siRNA. By contrast, surface expression of ESL-1 is reduced in GBF1-depleted cells. Bars, 16 µm. (H) HeLa cells were untreated (`con'; control) or transfected with scrambled siRNA (`scr') or siRNA against GBF1 (`siRNA') for 72 hours. Cells were lysed, and lysates were western blotted with the indicated antibodies. The level of ESL-1 is decreased in GBF1-depleted cells. (I,J) HeLa cells were transfected with scrambled siRNA (`scr') or with siRNA against GBF1 (`siRNA'). After 72 hours, cells were labelled with 35S-Met/Cys for 30 minutes and chased for the indicated times. At each time point, cells were lysed. Lysates were mock treated or treated with Endo-Hf, and ESL-1 was precipitated and analysed by SDS-PAGE and fluorography. In scr cells, immature ESL-1 (red arrowhead) is processed to a terminally glycosylated Endo-H-resistant form (green arrowhead). In GBF1-depleted cells, ESL-1 remains in the immature form (red arrowhead). The levels of ESL-1 remaining during the chase were quantitated and presented as the ESL-1 present at 1 hour of chase expressed as a percentage of that present at 0 hours of chase. The data show significant degradation of ESL-1 in GBF1-depleted cells.
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