Fig 2.

FXR physically interacts with ChREBP and HNF4α in vitro. (A) ChREBP protein levels in nuclear extracts from IHH incubated for 24 h in a medium containing low (1 mM) or high (11 mM) glucose concentrations and vehicle (DMSO) or GW4064 (5 μM). The expression of ChREBP protein was analyzed by Western blotting using a specific antibody. (B) In vitro GST pulldown experiments using full-length GST-FXR and in vitro transcribed/translated (TNT) ChREBP, Mlx, or HNF4α in the presence of [³⁵S]methionine. (C) In vitro GST pulldown experiments using GST (lane 2) and the indicated FXR deletion mutant protein (lanes 3 and 4) and TNT ChREBP in the presence of [³⁵S]methionine and vehicle (DMSO) or FXR ligand (GW4064, 5 μM). AF-1, activation function 1; DBD, DNA binding domain; LBD, ligand binding domain. (D) Two distinct anti-FXR antibodies were used for coimmunoprecipitation of ChREBP in nuclear extracts from IHH cells transfected with pSG5-FXR, pCMV-Sport6-ChREBP, and pcDNA3-Mlx and incubated in a medium containing a high (11 mM) glucose concentration. The expression of FXR and ChREBP proteins was detected 24 h after transfection by Western blotting (WB) using specific antibodies (AB1 and AB2).