FIGURE 2.

In vitro SUMOylation of FXR. Recombinant wild-type FXR and the various FXR mutant proteins were generated to identify FXR sites subject to SUMOylation. The in vitro SUMO assay contains the specified form of FXR along with the SUMO conjugation machinery (using the Active Motif Kit). Proteins were separated by SDS-PAGE and the conjugated protein detected by both the anti-Sumo1 and anti-FXR antibodies. Point mutations were made in Lys¹²² and Lys²⁷⁵ (replaced with arginine) with or without mutation of an adjacent Glu²⁷⁷ (replaced with alanine) or all three amino acid residues were mutated together, and then the recombinant FXRs subjected to in vitro SUMOylation. The K122R, K275R, and E277A single mutants were also SUMOylated by Sumo1. Mutation of two SUMO consensus sites (K122R, K275R, and E277A together) completely abrogated the in vitro SUMOylation of FXR. (lane 6). A control reaction contained the unconjugatable Sumo1 mutant protein provided by the SUMOlink kit or no FXR protein (lanes 7 and 8).