FIGURE 2.

Accumulation of complexes of C/EBPβ and HDAC1 in DEN-treated mice. A, levels of C/EBPβ, HDAC1, and C/EBPβ-HDAC1 complexes are increased in DEN-treated mice. Western blotting was performed with nuclear extracts isolated at different time points after DEN injection. Wks, weeks. B, levels of C/EBPβ and HDAC1 mRNAs are not changed during development of liver cancer. Quantitative RT-PCR was performed with RNA isolated from livers at different time points after DEN injection. C, C/EBPβ-HDAC1 complexes are elevated 6–25 weeks after DEN injection. HDAC1 was immunoprecipitated from nuclear extracts, and C/EBPβ was examined after immunoblotting of these immunoprecipitates. D, examination of HDAC1 by two-dimensional gel electrophoresis. Nuclear extracts from control livers (Con) and livers from DEN-treated mice were separated by a two-dimensional technique and probes with Abs to HDAC1. DEN + CIP, proteins were treated with alkaline phosphatase prior to two-dimensional separation. E, dephosphorylation of HDAC1 reduces formation of C/EBPβ-HDAC1 complexes. Protein extracts from 6 and 10 weeks of DEN-treated mice were incubated with CIP and used for co-IP and Western blot analyses. C/EBPβ was immunoprecipitated, and HDAC1 was examined in these immunoprecipitates. Data for two animals for each time point are shown.