Figure 1. Identification of FXR-associated cofactors.

(A) Representative silver-stained acrylamide gel showing polypeptides from HepG2 extracts specifically bound to unliganded FXR LBD (–GW4064) or liganded LBD (+GW4064). Arrowheads indicate excised bands processed for mass spectrometry analysis. Selected proteins yielded a peptide coverage above 20% and were detected at least twice out of 3 analyses. (B) Detected interactions with FXR. Interactions validated by GST pull-downs are indicated here. Solid line: ligand-dependent interactions; dotted line: constitutive interactions. (C) FXR interacts with both AMPKα1 and AMPKγ1. GST pull-down experiments were carried out using GST-fused flFXR (GST-FXR) or FXR LBD (GST-FXR LBD) and ³⁵S-labeled AMPKα1 and AMPKγ1. (D and E) AMPK coimmunoprecipitates with FXR. HepG2 cells were treated with 2 μM GW4064 and/or 1 mM AICAR for 2 hours. (D) Western blot analysis of whole-cell extracts was performed to assess the presence of the indicated proteins. (E) Whole-cell extracts were immunoprecipitated with either a nonspecific IgG or an anti-FXR antibody. Immunoprecipitated material was analyzed by Western blot using the indicated antibodies. Western blot analysis was carried out to assess AMPK and FXR expression levels in whole cell extracts (upper panel). (F) AMPK coimmunoprecipitates with FXR in mouse liver extracts. Mice received the indicated treatment (see Figure 6 for details), and FXR was immunoprecipitated as in E.