Fig 1: IFT172 exhibits ubiquitin conjugation activity in the presence of UbcH5a.(A) Western blot analysis of an in vitro ubiquitination assay containing Mus musculus Ube1 (E1), HsUbcH5a (E2), Ubiquitin (Ub), and ATP in the presence of various C-terminal IFT172 constructs as potential E3 ligases. Reactions were visualized by immunostaining with anti-Ubiquitin antibody. (B) Western blot analysis of in vitro ubiquitination assays containing Ube1 (E1), Ub, ATP, and HsIFT172C1, in the presence of 11 different ubiquitin-conjugating E2 enzymes (Abcam #ab139472). Reactions were visualized by immunostaining with (top) anti-Ubiquitin antibody and (bottom) Coomassie staining. Reactions were conducted under non-reducing conditions, accounting for the visualization of significant amounts of E1~Ub conjugate in the blot. (C) In vitro ubiquitination assays containing M. musculus 6XHis-Ube1, 6XHis-TEV-HsUbcH5a, Ub, and 6XHis-TEV-HsIFT172C1. Reactions were visualized by immunostaining with an anti-His tag antibody. (D) Western blot analysis of in vitro ubiquitination reactions containing M. musculus Ube1 (E1), HsUbcH5a (E2) WT/C85S mutant, Ubiquitin (Ub), HsIFT172C1, and ATP. As specified in each reaction, a reaction component was omitted (indicated by Δ) or a mutant component was used instead of the corresponding WT component (indicated by +). Reactions were visualized by immunostaining with (top) anti-Ubiquitin antibody and (bottom) Coomassie staining. (E) Analysis of the putative E2 binding site in the (center) HsIFT172 U-box domain and (left) U-box domain of ScPRP19. Both U-box domains are shown facing the E2 binding site. The PRP19 residues I5, Y31, and P39 whose mutagenesis leads to the loss of its ubiquitin ligase activity are represented as sticks (also highlighted in boxes within the sequence alignment in Figure 2E). The equivalent residues in the putative E2 binding site of IFT172 are also depicted as sticks. (Right) Surface amino acid conservation map for the E2 binding site in IFT172 U-box domain, color-coded as indicated. (F) Western blot analysis of in vitro ubiquitination assay with HsIFT172C1 WT and the specified HsIFT172C1 U-box variants. Reactions were visualized by immunostaining with (top) anti-Ubiquitin antibody and (bottom) Coomassie staining. Figure 3—source data 1.Uncropped labelled blot and Coomassie images (anti-Ubiquitin and anti-His western blots plus Coomassie-stained gels) underlying the in vitro ubiquitination assays shown in Figure 3A–D and F. Figure 3—source data 2.Original unprocessed raw image files of the blots, anti-Ubiquitin and anti-His western blots, and Coomassie-stained gels underlying Figure 3A–D and F.