Reduction of BMP signaling and hepcidin expression in neogenin mutant livers and in mutant hepatocyte culture. (A) Western blot analyses of liver lysates derived from wild-type (+/+) and neogenin mutant (m/m) mice during development using indicated antibodies; P3 indicates postnatal day 3. Note that the up-regulation of pSmad1/5/8 after P12 was blocked in neogenin mutant mice. (B) Quantification analysis of data from panel A. Three different mice from each age group were analyzed. Mean ± SEM is shown (n = 3). *P < .01, significant difference from the wild-type control. (C-E) Real-time PCR analysis of the expression of Id1, Smad7, and Atoh8 in wild-type and neogenin mutant livers. The expression was normalized to the reference gene NADPH mRNA. Mean ± SEM shown (n = 3). *P < .01, significant difference from the wild-type control. (F) Western blot analysis of lysates of primary cultured hepatocytes derived from wild-type and neogenin^m/m mice. Primary hepatocytes cultured for 5 days were treated with BMP2 (100 ng/mL) for the indicated times. Equal amounts of total proteins were immunoblotted with the indicated antibodies. (G) Quantitative analysis of data from panel A. Mean value was shown, which was from 2 independent experiments. (H-I) Real-time RT-PCR analysis of BMP2 induced (H) and basal (I) hepcidin expression in primary hepatocytes and in livers (D) derived from wild-type and neogenin mutant mice. Primary hepatocytes isolated from perfused livers of wild-type (C57BL/6) and neogenin mutant mice were cultured in collagen coated 12-well plates for 2 hours and treated with BMP2 (100 ng/mL) for 15 hours. The expression of hepcidin and NADPH (as a normalization gene) was assayed by quantitative real-time RT-PCR using TagMan probes. All samples were processed in triplicate, and a graph of mean values ± SEM from 3 independent experiments is shown. *P < .01, significant difference from wild-type control (t test).