Lysosomal Degradation Sequence Figure 3. Effect of endosomal lipid dynamics on RhoB distribution. (a) BAEC were treated with vehicle or 10 mM U18666A for 24 h, fixed and analyzed by transmission EM. Bars, 1 mm. (b) Confocal microscopy GFP-RhoB-transfected cells treated as in A. DIC, fluorescent image and their overlay are shown. Bars, 20 mm. (c) BAEC were transfected with the indicated constructs and treated with vehicle or U18666A; (a,b) and (e,f), confocal microscopy of live cells incubated with 25 nM LTR; cell contours are shown by dotted lines in (e,f); (c,d) distribution of LBPA observed by IF. Nuclei were stained with DAPI. Bars, 10 mm. (d) The degree of internalization of GFP-RhoB or GFP-Rab7 into U18666A-induced structures was estimated from co-localization of the corresponding constructs with LTR and it is shown as average values 6 SEM from three independent experiments (*p,0.05 vs control). (e) BAEC transfected with GFP-RhoB and treated with U18666A were analyzed by IEM with anti-GFP antibodies. Bar, 0.5 mm. (f) Effect of anti- LBPA on RhoB distribution. BAEC transfected with GFP-RhoB were incubated with a control antibody or with anti-LBPA (hybridoma supernatant diluted 1:1 in culture medium) for 20 h; (a,b) live cells observed by confocal microscopy; (c,d) accumulation of LBPA observed by IF with anti-mouse Ig antibody. Bars, 20 mm. (g) Control or NPC fibroblasts were transfected with GFP-RhoB. Co-localization with LTR was assessed in live cells. Bars, 20 mm. Co-localization is shown as average values 6 SEM of determinations from three experiments (*p,0.05 vs control). doi:10.1371/journal.pone.0008117.g003 PLoS ONE | www.plosone.org 5 December 2009 | Volume 4 | Issue 12 | e8117