Figure 4.

GRASP65 depletion does not perturb protein traffic along the secretory pathway. (A) The localization of KDEL-receptor, a marker of protein traffic between ER and the early Golgi, was analyzed in HeLa cells 48 h after transfection with scrambled control or GRASP65-specific siRNA. (B) The transport of the vesicular stomatitis virus G-protein (VSV-G) was analyzed in control and GRASP65-depleted cells. Cells were transfected with scrambled control or GRASP65-specific siRNA, followed by the transfection with a plasmid encoding for VSV-G (tsO45)-GFP. After a 24-h incubation at 37°C, the cells were shifted to 40°C for 8 h to arrest VSV-G in the ER. Cells were then shifted to the permissive temperature of 32°C to allow progression of VSV-G-GFP along the secretory pathway. Samples were taken at the indicated time and fixed for immunofluorescence microscopy to localize VSV-G-GFP and GRASP65, respectively.