Lysosomal Degradation Sequence Figure 5. Structural determinants involved in the subcellular localization and degradation of GFP-RhoB. (a,b) NIH-3T3 fibroblasts were transiently transfected with the indicated GFP constructs. 24 h after transfection, the distribution of fluorescent constructs was assessed by confocal microscopy of live cells. Bars, 20 mm. The putative posttranslational modifications of each construct are shown schematically above the panels. Construct stability was assessed by blocking protein synthesis with CHX and estimating the remaining levels by Western blot with anti-GFP antibody. Blots show the levels of the constructs of interest and of RhoGDI, as control. Lower panels show the construct levels remaining after 6 h treatment with CHX. wt, GFP-RhoB; C193S, GFP-RhoB C193S; C189S, GFP-RhoB C189S; C192S, GFP-RhoB C192S; C189,192S, GFP-RhoB C189,192S; RhoA, GFP- RhoA. Results are shown as average values 6 SEM of three different experiments (*p,0.05 vs wt levels in (a) and vs GFP-4 levels in (b)). doi:10.1371/journal.pone.0008117.g005 PLoS ONE | www.plosone.org 8 December 2009 | Volume 4 | Issue 12 | e8117