LDN-193189 and soluble hemojuvelin protein (HJV.Fc) block Smad1/5/8 signaling and inhibit Hamp mRNA expression in primary rat hepatocytes. Primary rat hepatocytes were isolated from female Lewis rats and stimulated with BMP6 (25 ng/mL; 0.69nM) for 12 hours in the presence/absence of LDN-193189 (500nM) or HJV.Fc (25 μg/mL; 166nM). (A) Quantitative RT-PCR for Hamp mRNA expression relative to the housekeeping transcript β glucuronidase (Gusb) was then carried out. (B) In parallel, Western blots investigating Smad1 levels and Smad1/5/8 phosphorylation (pSmad1/5/8) as well as Stat3 levels and phosphorylation (pStat3) were carried out. 1TBP18 was used as nuclear loading control. (A) Results are reported as means ± SEM for 3 independent experiments with n = 6 per group, and the P values are shown as determined by ANOVA with Bonferroni correction for multiple tests. (B) One representative blot of 3 independent experiments is shown.