Fig. 2: Ufmylation of CYB5R3 on the ER.
From: The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3
a Immunoprecipitation assay. HEK293T expressing UFL1 and UFBP1 were fractionated into microsomal (M) and cytoplasmic (C) fractions. The fractions were denatured with TNE with SDS (final 1%), diluted 10-fold with TNE, and then immunoprecipitated with CYB5R3 antibody followed by immunoblot analyses. Data shown are representative of three separate experiments. Bar graphs show the relative value of the ratio of endogenous ufmylated CYB5R3 to CYB5R3 was 1 when neither overexpression nor knockdown of any E3 components was performed. Data are means ± s.e. Statistical analysis was performed by two-sided Welch’s t test. b Immunoprecipitation assay. UFL1 or UFBP1 in UFSP2−/− HEK293T were knocked down, and the cells were fractionated into microsomal (M) and cytoplasmic (C) fractions. The fractions were analyzed as described in a. Data shown are representative of four separate experiments. Bar graphs show the relative value of the ratio of endogenous ufmylated CYB5R3 to CYB5R3 was 1 when neither overexpression nor knockdown of any E3 components was performed. Data are means ± s.e. Statistical analysis was performed by Šidák’s multiple comparison test after one-way ANOVA. c Immunoblot analysis. The indicated constructs were transfected into CYB5R3−/− UFSP2−/− HEK293T. 48 h after transfection, the cell lysates were fractionated into microsomal (M) and cytoplasmic (C) fractions followed by immunoblot analysis. Data shown are representative of three separate experiments. Asterisk indicates a degradative product of CYB5R3-His-3xFLAG. Since the CYB5R3 in the cytoplasmic fraction (indicated by asterisk) is almost as mobile as ΔN26, it is considered to be the N-terminal truncated form. d Immunofluorescence analysis. GFP-tagged wild-type (CYB5R3-GFP) or ER-localization-defective CYB5R3 (CYB5R3ΔN26-GFP) were transfected into CYB5R3−/− HeLa. 48 h after transfection, the cells were immunostained with anti-PDI antibody. Bars: 10 μm. Data shown are representative of three independent experiments. Source data are provided as a Source Data file.
