Fig. 2.

The localisation of UBL3 to MVBs depends on UBL3 modification. a Representative projected images of MDA-MB-231 cells transfected with EGFP-UBL3 and co-stained with markers for MVB (CD63, n = 10), early endosome (EEA1, n = 10), lysosome (LysoTracker, n = 10), or recycling endosome (Rab11, n = 10). The regions in the dotted box are shown as a single confocal image in the inset. Scale bars, 10 and 1 μm. b Quantitative analysis of EGFP-UBL3 fluorescence intensity in a. *, p < 0.05; ***, p < 0.001 by Kruskal–Wallis/Dunn´s multiple-comparisons test. c Representative images of MDA-MB-231 cells transfected with EGFP-UBL3 (wild-type, n = 5; mutants, n = 5) and co-stained with CD63 values shown as % of total UBL3. Scale bars, 10 and 1 μm. d Immune-EM images of wild-type UBL3 and UBL3Δ1 in MDA-MB-231 cells. Scale bars, 500 nm (left and right panels) and 200 nm (middle panel). e Quantification of the numbers of gold colloids per area in d. MVB, n = 10; Plasma membrane, n = 10. *, p < 0.05; ***, p < 0.001 by two-tailed Student’s and Welch’s t-tests