FIG. 8.

BRE-1 and I-BRE respond to different concentrations of BMP-7. (A) ChIP of Smad1 bound to I-BRE and BRE-1. HepG2 cells were treated for 1 h with increasing concentrations of BMP-7. Protein-DNA complexes were cross-linked by formaldehyde treatment, cells were lysed, and DNA was sheared by sonication. Cell lysates were subjected to immunoprecipitation (IP) with an anti-Smad1 antibody (αSmad1 Ab). DNA recovered from the immunoprecipitation was quantified by real-time PCR. Diagrammed representations of PCR products are presented in Fig. 3A and 4A. PI, preimmune; −, absent. (B) The BRE-1 and I-BRE luciferase reporter constructs were transiently transfected in HepG2 and Neuro-2A cells in the presence or absence of 0.03 μg of Smad1 and Smad4 cDNA/well. Cells were incubated overnight with increasing concentrations of BMP-7. The relative luciferase activities of cell lysates were determined. (C) Smad7 expression is induced by different concentrations of BMP-7 in HepG2 and Neuro-2A cells. RNA was extracted from HepG2 (black bars) and Neuro-2A (gray bars) cells treated for 1 h with increasing concentrations of BMP-7. The RNA was reverse transcribed, and cDNA for Smad7 was quantified by real-time PCR. Levels of hypoxanthine phosphoribosyltransferase cDNA were used to normalize the results.