Fig. 1: Proteomic changes induced by knockdown of E2 ubiquitin-conjugating enzymes in human cells.

E2 knockdown in HEK293T cells was obtained via siRNAs for individual E2s or for groups of related E2s with high sequence homology (e.g. UBE2D1/2/3), which were targeted together by pooling E2-specific siRNAs. E2 siRNAs reduce (green) the mRNA (a) and protein (b) levels of the targeted E2s (boxed) specifically, i.e. without affecting other E2s, compared to non-targeting (NT) siRNAs. Significant (P < 0.05; unpaired two-tailed t test) transcriptional changes are highlighted in bold red (downregulated) and blue fonts (upregulated). The extent of downregulation is displayed in green shades whereas non-significant changes are shown in gray. c Deep-coverage TMT mass spectrometry identifies the proteome subsets that are regulated by E2 RNAi. Compared to NT siRNAs, E2 RNAi leads to significant protein upregulation (blue) and downregulation (red) (P < 0.05; Log₂FC > 0.2 and <–0.2) that do not arise from corresponding changes in mRNA levels, defined by RNA-seq. This data represents 6 sets of 16-plex TMTs of E2 siRNAs (n = 3 biological replicates/group), each with its own control NT siRNAs (n = 4 biological replicates). On average, each TMT set quantified 10700 proteins: 5132 of these (mapping to 4676 DAVID IDs) were modulated by ≥1 E2s. d JUMPptm analyses identify linkage-specific ubiquitin modifications modulated by E2s. In particular, the knockdown of many E2s reduces K48-linked ubiquitination whereas this increases upon heat shock. Other linkage-specific modifications (K6, K11, K27, K29, K33, and K63) are affected by fewer E2s. Significant downregulation (P < 0.05) of these modifications by E2 RNAi is displayed in bold fonts and shades of red whereas non-significant changes (P > 0.05) are shown in gray. Supplementary Fig. 3 reports the precise P values (one-way ANOVA). e Proteins that are regulated by E2 RNAi without significant mRNA changes are reported on the y-axis whereas the number of E2s regulating each protein subset is reported on the x-axis. Several GO categories are enriched among protein sets that are modulated by 5–9 E2s (f), 2–4 E2s (g), and by single E2s (h). Source data are provided in the Source data file.