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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: Hepatology. 2013 Feb 15;57(3):1098–1106. doi: 10.1002/hep.26146

Figure 4. FXR-mediated inhibition of gankyrin requires C/EBPβ and C/EBPβ-HDAC1 complexes.

Figure 4

A. Amounts of C/EBPβ-HDAC1 complexes are increased in cells with activated FXR. Input: Western blotting with nuclear extracts used for Co-IP studies. C/EBPβ-IP: C/EBPβ was immunoprecipitated from nuclear extracts and the IPs were probed with Abs to HDAC1 and C/EBPβ. Ag; agarose beads were incubated with nuclear extracts. CRM; cross-reactive molecule, serves as a loading control. B. Activation of FXR in Hepa 1-6 cells leads to the accumulation of the C/EBPβ-HDAC1 complexes on the gankyrin promoter. ChIP assay was performed with chromatin solutions from Hepa 1-6 cells treated with DMSO, CDCA and GW4064. In; 1/100 of input, B; beads. C. Expression of shRNA to C/EBPβ in stable clones inhibits C/EBPβ. Western blotting was performed with antibodies to C/EBPβ. Positions of full length (FL), LAP and LIP isoforms of C/EBPβ are shown by arrows. Bar graphs show the level of inhibition of C/EBPβ. D. C/EBPβ is required for the FXR-dependent inhibition of gankyrin. Gankyrin was examined in control Hepa 1-6 cells (NTG) and in stable clones of Hepa 1-6 cells with inhibited C/EBPβ. Bar graphs show ratios of gankyrin to β-actin. E. Knock-down of C/EBPβ in livers of WT mice activates expression of gankyrin. Expression of C/EBPβ and HDAC1 was examined in livers of mice were treated with control RNA (NTG) and with siRNA to C/EBPβ. Bottom image: C/EBPβ was immunoprecipitated from nuclear extracts and HDAC1 was examined in C/EBPβ IPs. F. Expression of gankyrin and C/EBPβ mRNAs in livers of siRNA-treated mice.