Fig. 2. The network of SENP5 interactors and targets.
a Volcano plot of quantitative MS analysis after anti-FLAG-IP of HEK923T cell lysates transiently expressing FLAG-SENP5C713S. Significantly enriched interactors are color-coded (log2 ratio ≥ 1, –log10 p value ≥ 1.3). Identification of candidates is based on two-sided Student’s t-test analysis comparing LFQ intensities of IPs from FLAG-SENP5 expressing and control cells. Experiments were performed as triplicates. b Volcano plot summarizing the results of a quantitative MS analysis comparing SUMO targets of HeLa cells transfected with siSENP5 or siControl. Proteins were enriched by anti-SUMO2/3 IP. Hits considered as significant are highlighted in red (log2 ratio ≥ 0.58, –log10 p value ≥ 1.3). Candidate SENP5 target proteins were defined by two-sided Student’s t-test analysis comparing LFQ intensities of anti-SUMO2/3 IP with the respective IgG control IPs. The experiment was performed with four replicates. c Venn diagram representing the overlap of proteins identified as SENP5 interactors (a) or SENP5 targets (b). d High-confidence SENP5 targets and interactors were subjected to GO BP analysis (interactors: log2 ratio ≥ 1, targets: log2 ratio ≥ 0.58). Displayed here are 8 out of the top 10 enriched processes. Highlighted in red are terms connected to ribosome biogenesis. The enrichment analysis was done using the ShinyGO tool, applying an FDR cutoff of 0.05. e STRING network analysis of significantly enriched interactors and targets of SENP5 (a, b). Ribosomal proteins or components of the SSU processome are highlighted in green or blue, respectively. Only connected proteins are visualized. f HEK293T cells were transfected with FLAG-tagged SENP3, SENP5 or empty vector control for 48 h. To stabilize short-lived SENP5, cells were treated with MG-132 (25 µM) or DMSO for 4 h prior to IP. Immunoblotting was performed as indicated. Input samples are shown in the left panel. Probing against the FLAG-tag was used to control the expression of the FLAG-tagged SENP3 or SENP5 constructs. g HeLa cells were transfected with siRNAs against SENP3, SENP5, SENP3 + SENP5 or siControl. 72 h post transfection, endogenous SUMO2/3 or IgG control IP were performed and samples were analyzed by immunoblotting as indicated. Input samples are shown in the left panel. Source data for (f) and (g) are provided as a Source Data file.