Figure S6 Levels of RhoGDI in BAEC detected by IF before and after CHX treatment. BAEC were treated as in Fig. 4, and fixed. Endogenous RhoGDI (in red) was detected by IF. Nuclei were stained with DAPI. Images shown are overall projections. Bars, 20 mm. Found at: doi:10.1371/journal.pone.0008117.s006 (4.04 MB TIF) Figure S7 Effect of inhibitors of isoprenylation and palmitoyla- tion on the levels of endogenous RhoB protein or of RhoB constructs in different cell types. (a) NIH-3T3 fibroblasts were transiently transfected with GFP or with GFP-RhoB, and construct levels were assessed by Western blot with an anti-GFP antibody after 24 h treatment with simvastatin (Sv) or 2-bromopalmitate (2- BP). (b) NIH-3T3 fibroblasts or BAEC were treated as in (a) and the levels of the indicated proteins were assessed by Western blot. Dotted lines show sites where lanes from the same gel have been cropped. (c) BAEC were transiently transfected with GFP-RhoB, treated with simvastatin or 2-BP for 3 h and visualized by live confocal microscopy. Note that in simvastatin-treated cells, GFP- RhoB can be detected in the nucleus, which is indicative of the presence of non isoprenylated construct. In contrast, in 2-BP- treated cells the appearance of GFP-RhoB is more diffuse than in control cells but the nucleus is excluded, as expected from an isoprenylated construct. Bars, 20 mm. Found at: doi:10.1371/journal.pone.0008117.s007 (3.78 MB TIF) Figure S8 Cellular distribution and stability of GFP-RhoGDI and GFP-RhoGDI-8. (a) BAEC were transiently transfected with the indicated constructs as in Figure 6, treated with 10 mM chloroquine for 24 h and incubated with 25 nM LTR for 15 min before observation of live cells by confocal microscopy. Bar, 20 mm. (b,c) NIH-3T3 fibroblasts were stably transfected with the indicated constructs. Protein stability (b) and subcellular localiza- tion of constructs in control and chloroquine-treated cells (c) were assessed as described in Fig. 6, after 30 days in culture. Bar, 20 mm. Found at: doi:10.1371/journal.pone.0008117.s008 (6.51 MB TIF) Figure S9 Hypothetical model for the degradation pathway of RhoB and -CINCCKVL chimeric proteins. Isoprenylated and References 1. Schwartz AL, Ciechanover A (2009) Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol 49: 73–96. 2. Fader CM, Colombo MI (2009) Autophagy and multivesicular bodies: two closely related partners. Cell Death Differ 16: 70–78. 3. Mellor H, Flynn P, Nobes CD, Hall A, Parker PJ (1998) PRK1 is targeted to endosomes by the small GTPase, RhoB. J Biol Chem 273: 4811–4814. 4. Huang M, Duhadaway JB, Prendergast GC, Laury-Kleintop LD (2007) RhoB regulates PDGFR-beta trafficking and signaling in vascular smooth muscle cells. 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