In vivo effect of heparin in mice. (A) Mice were daily treated subcutaneously for 7 days with 50 mg/kg/d UFH and the level of hepatic hepcidin mRNA evaluated by RT-PCR, HRPT1 was used for calibration, the corresponding real-time RT-PCR values were 2% and 2.7% of the control, for the mice treated with 50 mg/kg/d UFH. Bottom: Western blot of the liver homogenates for evaluation of phosphorylated SMAD1/5/8 and of total SMAD5. β-actin was used as load control. The real-time RT-PCR evaluation of Id1 mRNA in the treated mice is shown on the right. (B) Mice were treated subcutaneously for 15 days with the pharmacologic concentration of 2 mg/kg/d UFH or with saline, and liver hepcidin mRNA was evaluated by real-time RT-PCR of in relationship to HPRT1 mRNA. The animals were analyzed for spleen iron and for serum iron concentration. The horizontal bars represent the mean values.