Lysosomal Degradation Sequence Figure 2. Localization of RhoB along the endo-lysosomal pathway. (a) BAEC transiently transfected with GFP-RhoB were incubated with Texas Red-labeled dextran (Dextran-TXR) for 5 min and visualized by confocal microscopy after 1 h. Bar, 20 mm. (b) BAEC were transiently transfected with the indicated constructs and 24 h later live cells were observed by confocal microscopy. Bars, 15 mm. (c) BAEC were transfected with GFP-RhoB, RFP-RhoB and/or Lamp1-GFP and treated 24 h later with leupeptin plus E64d or chloroquine for further 24 h. When indicated, cells were incubated with 25 nM LTR for 15 min. Live cells were observed by confocal microscopy. Bars, 15 mm. (d) Transmission EM of MVB from control and chloroquine treated BAEC. Bars, 1 mm. (e) IEM analysis of GFP-RhoB in control and choroquine-treated cells by staining with anti-GFP antibody. Bars, 1 mm. doi:10.1371/journal.pone.0008117.g002 transfection efficiency, the presence of RhoB was assessed in transfected and non-transfected (NT) cells by IF. After CHX treatment, the proportion of RhoB positive cells was clearly reduced in the cases non-transfected (NT) cells and cells transfected with Vps4 wt but was preserved in cells transfected with Vps4 DN (Fig. 4c). Levels of RhoGDI did not decrease after PLoS ONE | www.plosone.org 4 December 2009 | Volume 4 | Issue 12 | e8117