Fig. 4: Ufmylated CYB5R3 interacts with UFBP1.

a CYB5R3^−/− UFSP2^−/− HEK293T expressing indicated constructs were lysed and then subjected to immunoprecipitation. b–c In vitro pull-down assay. The amount of each protein bound to GST-UFBP1 was estimated by immunoblot (b). The faster-migrating ufmylated CYB5R3ΔN26 proteins are most likely degradation products due to long-term storage and freeze–thawing. UFM1 and UFL1 binding to GST-UFBP1 and each mutant was estimated by immunoblot (c). d Top, analysis of the secondary structure of UFBP1 (116–214) using PSIPRED/DISOPRED^61,62 and COILS⁶³. Bottom, alignment of UFIM of UFBP1. e Crystal structure of UFM1 complexed with the UFIM of UFBP1 (left) or the UFIM of UBA5 (right, PDB 5HKH). The side chains of the residues involved in the UFM1-UFIM interaction are shown with a stick model. Broken lines indicate possible hydrogen bonds. f Structural comparison between UFBP1 and UBA5 UFIMs complexed with UFM1. The figure was prepared by superimposing the structure of the UFIM moiety of UFBP1-UFM1 onto that of UBA5 UFIM-UFM1. g In vitro pull-down assay. The amount of each protein bound to GST-UFBP1 was estimated by immunoblot. h–i Wild-type (h) and UFBP1^−/− HEK293T (i) expressing indicated constructs were lysed and then subjected to immunoprecipitation. j HEK293T expressing indicated constructs were lysed and then subjected to immunoblot. Bar graphs show the relative value of the ratio of endogenous ufmylated CYB5R3 to CYB5R3 was 1 and the relative value of the ratio of ufmylated UFBP1 to UFBP1 as 1 when UFL1 and UFBP1 were overexpressed, respectively. Data are means ± s.e. Statistical analysis was performed by two-sided Welch’s t test. k HEK293T expressing indicated constructs were lysed and then subjected to pull-down assay with Ni-NTA agarose under denaturing conditions, followed by immunoblot. Bar graph shows the relative value of the ratio of MYC-UFM1-conjugated CYB5R3-His-FLAG to CYB5R3-His-FLAG in crude lysates as 1 when UFL1 and UFBP1 were overexpressed. Data are means ± s.e. Statistical analysis was performed by Šidák’s multiple comparison test after one-way ANOVA. Data for immunoblots presented in this Figure are representative of three separate experiments. Source data are provided as a Source Data file.