HeLa cells expressing GFP alone or GFP-Rab protein fusions (green), were infected with Red- (blue)

Abstract

<p><b>Copyright information:</b></p><p>Taken from " Trafficking is Defined by Continuous Dynamic Interactions with the Endolysosomal System"</p><p></p><p>Traffic (Copenhagen, Denmark) 2007;8(3):212-225.</p><p>Published online 15 Jan 2007</p><p>PMCID:PMC2063589.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> Dextran-647 (red) was added 2 h p.i. to load the endocytic pathway. Live-cell imaging was initiated 1 h after addition of dextran-647. A single confocal plane is shown (D, overlay) to illustrate the presence of dextran-647 (B) in SCVs containing Red- (C) that are outlined by GFP-Rab7 wt (A). Scale bar = 5 μm. E) Quantification of dextran-647 and V-ATPase positive SCVs. SCVs containing dextran-647 (gray bars) were scored in GFP-expressing cells and expressed as percentage of dextran–647 positive SCVs in mock-transfected cells (set to 100%). For quantification of V-ATPase accumulation on the SCV, transfected cells (black bars) were infected with Red- then fixed and processed for immunofluorescence 1 h p.i. V-ATPase was detected using mouse monoclonal antibodies followed by Cy5-conjugated donkey α-mouse antibodies. SCVs staining positive for V-ATPase were counted in GFP-expressing cells and expressed as percentage of V-ATPase positive SCVs in mock-transfected cells (set to 100%). Values are the mean ± S.D. of three independent experiments ( ≥ 50 in each experiment). Asterisk indicates significant difference from all other conditions (p < 0.05, , Tukey’s test)