Lysosomal Degradation Sequence Figure 4. Effect of Vps4 wt and DN on RhoB localization and stability. (a–b) BAEC were transfected with GFP-Vps4 wt or DN, as indicated, and 24 h later, treated in the absence or presence of 10 mM chloroquine for 24 h. In (b), 25 nM LTR was added 15 min before observation of live cells by confocal microscopy. Bars, 15 mm. (c) BAEC were transfected as above and 24 later placed in serum-free medium. After 24 h, control (0 time, -) cells were fixed, and the rest were treated with 20 mg/ml CHX for 8 h (+) to allow degradation of endogenous RhoB, before fixation. Endogenous RhoB (in red) was detected by IF. Nuclei were stained with DAPI. Images shown are overall projections. Bars, 20 mm. The lower panel shows the proportion of RhoB-positive cells among non-transfected (NT) cells or among cells transfected with the indicated constructs before (2) or after CHX (+) treatment. At least 40 cells of each type were monitored from four independent experiments. Results are shown as average values 6 SEM (*p,0.05 vs the same condition in the absence of CHX). (d) COS7 cells were transfected with GFP-Vps4 wt or DN, as indicated, and 24 h later split in three dishes for treatment with CHX for the indicated times. RhoB and RhoGDI levels were assessed by Western blot. Results shown are average values 6 SEM from three experiments (*p,0.05 vs GFP-Vps4 DN at the same time points). doi:10.1371/journal.pone.0008117.g004 PLoS ONE | www.plosone.org 6 December 2009 | Volume 4 | Issue 12 | e8117