Fig. 1: CYB5R3 is a bona fide substrate for UFM1.

a Immunoblot analysis. Wild-type or UFSP2^−/− HEK293T expressing UFL1 and UFBP1 were fractionated into microsomal (M) and cytoplasmic (C) fractions followed by immunoblot analysis. Data shown are representative of three separate experiments. b An experimental approach to identify ufmylated proteins. c Domain structure of CYB5R3. Membrane anchor, FAD-binding site, and lysine residue for UFM1 conjugation are indicated. d Immunoblot analysis. The indicated constructs were transfected into parental or UFSP2^−/− HEK293T. 48 h after the transfection, the cell lysates were pulled down with Ni-NTA agarose under denaturing conditions followed by immunoblot analysis. Data shown are representative of three separate experiments. e Immunoblot analysis. A series of CYB5R3KR mutants together with MYC-UFM1, MYC-UFL1, and UFBP-MYC were expressed in UFSP2^−/− HEK293T. The cell lysates were analyzed as described in d. Data shown are representative of three separate experiments. f Alignment of the region around K214 of CYB5R3. g In vitro conjugation assay. Recombinant UFM1, UBA5, and UFC1 from E. coli and FLAG-UFL1 and UFBP1-MYC from UFC1-knockout HEK293T cells were incubated with a microsomal fraction prepared from UFBP1-deficient HEK293T cells in the presence or absence of ATP for 90 min, and the mixture was subjected to SDS-PAGE followed by immunoblot analysis with CYB5R3 antibody. Data shown are representative of three separate experiments. h Immunoblot analysis. The indicated constructs were transfected into UFSP2^−/− HEK293T. 48 h after transfection, cell lysates were subjected to immunoblot analysis. Data shown are representative of three separate experiments. Source data are provided as a Source Data file.