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. 2014 Nov 25;34(2):184–199. doi: 10.15252/embj.201489527

Figure 3. SUMO2 modification of agonist-activated FXR at K277 represses NF-κB target inflammatory genes.

Figure 3

  • A Two consensus SUMO sites in FXR.
  • B, C In vitro SUMO assay: Flag-FXR-WT or FXR mutants were incubated with purified SUMO components as indicated, and SUMO2 modification of FXR was detected by IB.
  • D Alignment of the FXR region containing K277 from various species.
  • E, F In-cell SUMO assay: COS-1 cells were transfected with flag-FXR and with E3 SUMO ligase as indicated, and SUMOylated FXR was detected by IP/IB.
  • G In-cell SUMO assay: Flag-FXR-WT and siRNA targeting PIASy were expressed in hepatocytes isolated from lean mice and treated with GW4064 for 30 min, and SUMOylated FXR was detected by IP/IB.
  • H Mice were treated with GW4064 or fed chow containing 0.5% CA for 3 h, and SUMO2-modified endogenous hepatic FXR was detected by IP/IB. Veh: Mice fed a normal diet and treated with vehicle, DMSO.
  • I Mice were infected with Ad-FXR-WT or the K277R mutant and treated with GW4064, and SUMO2-FXR levels were detected by IP/IB. Values are presented as mean ± SEM (n = 3 mice).
  • J COS-1 cells were infected with adenoviral vectors and transfected with SUMO2 plasmid as indicated, and SUMO2-FXR levels were detected by IP/IB. The membrane was stripped and reprobed with FXR antibody.

Source data are available online for this figure.