Fig 2: Vps11/18 carry E3 ubiquitin ligase activities involved in signal transduction. a Scheme of the class C Vps proteins that constitute the core of the HOPS and CORVET complexes. The RING domain is highlighted in purple. b In vivo ubiquitination assay with HEK293T cells overexpressing the indicated Vps proteins along with HA-tagged ubiquitin. Ubiquitinated proteins were immunoblotted (IB) with an HA antibody; GAPDH is the internal loading control. c Experimental strategy to quantify the ubiquitinome upon overexpression of Vps11/18 in HEK293T cells using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry; see text for more details. d, e Volcano plots showing the differentially ubiquitinated sites (log2 ratios) between cells transfected with the empty expression vector and cells overexpressing either Vps11 (d) or Vps18 (e) and their statistical significance (−log10(p-value) on the y-axis); values above the black dotted line are significantly regulated; significantly downregulated sites (blue dots) are at the top left of the blue threshold line while significantly upregulated sites (red dots) are at the top right of the red threshold line. f Table of the number of protein substrates of Vps11/18 with one or several differentially regulated ubiquitination sites. g Venn-diagram of the differentially ubiquitinated proteins in Vps11- or Vps18-overexpressing cells compared to the empty vector condition (at the top) with the zoomed in shared proteins ordered by hierarchical clustering (at the bottom); arrows point to E3 ubiquitin ligases that are more ubiquitinated upon Vps11 or Vps18 overexpression. h GO-based clustering of the interactome of the proteins associated with a significant change of the ubiquitinome upon Vps11/18 overexpression. Each node represents a GO term with a color code showing the relative changes of the corresponding ubiquitinome upon Vps11 (inner circle) or Vps18 (outer circle) overexpression (blue or red for enriched in control cells over cells overexpressing Vps11/18 or the other way around, respectively). The number of proteins with a particular GO term is represented by the size of its corresponding node. Source data are provided as a Source Data file