Overexpression of GDP-locked Rab41 partially inhibited VSV-G transport from ER to Golgi apparatus.

<p>Wild type HeLa cells were microinjected with either 25 ng/µl plasmid encoding the tsO45 mutant of VSV-G-GFP (Control) or a mixture of 100 ng/µl myc-tagged GDP-locked Rab41 encoding plasmid and 25 ng/µl tsO45 mutant of VSV-G-GFP encoding plasmid. After 24-h incubation at 39.5° C, VSV-G was accumulated in the ER (A, upper panel). Cells were then shifted to 32° C, permissive conditions for VSV-G transport, and incubated for 40 min in the presence of cycloheximide to prevent further protein synthesis (A, lower panel). Cells were then fixed and visualized by wide field light microscopy. At the end of a 40-min chase, Golgi accumulation of VSV-G was observed in both control and GDP-locked Rab41 overexpressing cells. However, VSV-G retention in the ER for GDP-locked Rab41 overexpressing cells was much higher than that in control cells (A, lower panel, and B). Successful co-injection was confirmed by antibody staining. All images shown or used for quantification were single-plane deconvolved. Error bars represent the mean ± SEM of ~20 injected cells. Asterisks mark injected cells.