Ubiquitin-dependent internalization of Box-GFP-Ub. Expression of markers/reporters in tobacco mesophyll protoplasts as indicated below. A-C: Box-GFP-Ub (green) and the TGN/EE marker YFP-SYP61 (red) partially colocalize (arrowheads). D-F: Box-GFP-Ub (green) and MVB/LE marker Ara6-RFP (red) colocalize to a high degree (arrowheads). Box-GFP-Ub localizes to characteristic ring-like MVB structures after wortmannin-treatment (+WM; inset in D). For quantitative analysis of colocalization see Additional file 2. Endosomal signals of Box-GFP-Ub (G) are significantly reduced in quantity if coexpressed with the clathrin hub (RFP-Hub1; red, H). Arrowheads indicate remaining Box-GFP-Ub-positive endosomes. I: Statistical analysis reveals that the punctate signals are about 57% less abundant in cells coexpressing the RFP-Hub1 (+) as compared to control cells (−). (***) p <0.0001. Scale bars = 5 μm.