Figure 4.

Identification of protein binders for the free ubiquitin peptide by using PLATO-BC assay

A. Rank of ORF enrichment from the PLATO-BC assays for biotinylated ubiquitin (Bio-Ub) relative to free biotin (Bio). 14,145 ORFs were ranked according to the ratio of Bio-Ub/Bio (from the smallest to the largest). The red line shows the cut-off value. B. GO analysis of top-ranked ORFs was performed using GOrilla. Clustering analysis and visualization were performed using REVIGO. Circle size represents the uniqueness of the GO term in EBI GOA database (more general terms have larger circle size). Circle color indicates the P value. C. mRNA enrichment of a set of known ubiquitin-binding proteins from the PLATO-BC assay of Bio-Ub. mRNA level was quantified by RT-qPCR. Relative enrichment of ubiquitin-binding proteins was normalized to Bio. Data are presented as mean ± SD (n = 3). ***, P < 0.001; ****, P < 0.0001, ANOVA. D. Interaction of RIOK3 with ubiquitin in vitro. pET-DEST42-RIOK3 was transformed into the E. coli DE3 strain. V5-His6-tagged RIOK3 protein was purified and subjected to protein pull-down assays for Bio-Ub or Bio. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-V5 antibody. E. The preferable interaction of RIOK3 with K63-polyubiquitin chains. pcDNA-DEST43 was co-transfected into HEK293T cells with the vector expressing HA-tagged wild-type (wt), K63- or K48-specific ubiquitin. Cell lysate was prepared and subjected to the protein immuno-precipitation assays for V5-His6-tagged RIOK3 using an anti-V5 antibody or a mouse IgG (mIgG) control. Protein samples were separated by SDS-PAGE and analyzed by immunoblotting using an anti-HA antibody. GO, gene ontology; RIOK3, RIO kinase 3; Ub, ubiquitin.