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. 2023 Apr 3;14:1840. doi: 10.1038/s41467-023-37362-7

Fig. 4. DNA damage induces FBF1-UBC9 association and FBF1 SUMOylation, which are negatively regulated by the ARL13B-ARL3 GTPase cascade.

Fig. 4

a In vitro SUMOylation assay of FBF1 and western blot using a SUMO1 antibody. GST tagged 598–1148 domain FBF1 was purified using E. coli. b Endogenous UBC9 immunoprecipitates with FBF1 in RCTE cells at day 7 after irradiation. c Endogenous FBF1 immunoprecipitates with SUMO1 in RCTE cells at day 7 after irradiation. d Endogenous UBC9 immunoprecipitates with ARL3 in RCTE cells. e GST pull-down assay shows the ARL3 and UBC9 interaction. f Endogenous FBF1 immunoprecipitates with UBC9 in WT or ARL3-/- RCTE cells at day 7 after irradiation. g Endogenous FBF1 immunoprecipitates with SUMO1 in WT or ARL3-/- RCTE cells with or without IR treatment. h ARL3 inhibited the interaction between FBF1 and UBC9 by co-immunoprecipitation in 293T cells. i ARL3 reduced the interaction between FBF1 and UBC9 by GST pull-down assay. GST-tagged C-terminal fragment (598-1148) of FBF1, His-ARL3 and His-UBC9 were purified using E. coli. j In vitro SUMOylation assay was performed in a reaction with GST-tagged C-terminal fragment (598-1148) of FBF1 and His-ARL3 purified in E. coli. Western blot was detected by SUMO1, GST and His antibody. k Endogenous UBC9 immunoprecipitates with Myc-ARL3 or FBF1 in ARL3WT, ARL3DA or ARL3DN overexpression RCTE cells at day 7 after irradiation. Three experiments were repeated independently with similar results (a–k). Source data are provided as a Source Data file.