Lysosomal Degradation Sequence Figure 7. Subcellular localization and stability of chimeric proteins bearing -CINCCKVL C-terminal sequences. (a) BAEC were co- transfected with tagRFP or tagRFP-CINCCKVL (tagRFP-8) and GFP-RhoB or Lamp1-GFP, as indicated, treated for 24 h in the absence or presence of 10 mM chloroquine and directly visualized by confocal microscopy. Images shown are individual confocal sections. Bars, 20 mm. (b) The stability of tagRFP and tagRFP-8 was assessed in BAEC as described in Fig. 5, using an anti-tagRFP antibody. Results are shown as average values 6 SD of at least three determinations (*p,0.05 vs tagRFP levels at the same time points). (c) BAEC were transfected with GFP-RhoGDI, GFP-RhoGDI-8 and/or RFP- RhoB, incubated with 25 nM LTR, as indicated, and directly observed by confocal microscopy. Bar, 20 mm. (d) BAEC were transfected with GFP- RhoGDI-8 in the absence (a) or presence (c) of 10 mM simvastatin. 24 h after transfection, confocal images of live cells were obtained (a, c). At that point, simvastatin was withdrawn from (c) and added to (a). After a 24 h additional incubation images of live cells were taken (b, d). Bar, 20 mm. (e,f) BAEC were transiently transfected with GFP-RhoGDI-8 or GFP-RhoGDI. In (e) protein synthesis was blocked by CHX addition. In (f) BAEC were treated with the indicated agents for 24 h. Levels of GFP-RhoGDI-8, GFP-RhoGDI and endogenous RhoGDI, were assessed by Western blot. Sv, simvastatin; Chlo, chloroquine; Leu, leupeptin. doi:10.1371/journal.pone.0008117.g007 PLoS ONE | www.plosone.org 10 December 2009 | Volume 4 | Issue 12 | e8117