Fig 2: Effect of iron status on mouse liver Hamp, Bmp6, Tmprss6 and Id1 mRNA. Hamp, Bmp6, Tmprss6 and Id1 mRNA was determined by real-time PCR and expressed relative to β-actin mRNA. C: Control, Fe: Iron injection (200 mg/kg), ID: Iron-deficient diet, Bl: Bleeding (0.6 ml of blood once weekly for 3 weeks), Epo: Erythropoietin (50 U/day) administration for 4 days, WT: Hjv+/+ mice, KO: Hjv−/− mice. Asterisks indicate statistically significant difference (n = 4, p<0.05).

Fig 3: Effect of iron status on rat liver membrane hemojuvelin.Immunoblots of Hjv protein in rat liver crude membrane fractions. Panel A: Detection with primary anti-Hjv antibody AF3634. Panel B: Identical samples detected with primary anti-Hjv antibody AF3720. Panel C: Densitometric quantification of the 35 kDa Hjv band. Liver samples from Hjv+/+ and Hjv−/− mice are included for comparison. C: Control; Fe: Iron injection (1200 mg/kg); ID: Iron-deficient diet. WT: Hjv+/+ mice, KO: Hjv−/− mice. 60 µg of protein was loaded per lane. Arrows indicate Hjv-specific bands. Connexin 43 was used as loading control. For densitometry, the optical density of control samples was set at 100%, n = 3.

Fig 4: Determination of mouse plasma hemojuvelin by immunoblot and ELISA.Panel A: Immunoblot of untreated mouse plasma (80 µg of protein per lane). Panel B: Immunoblot of albumin-depleted mouse plasma (60 µg of protein per lane). Primary anti-Hjv antibody: AF3720. Panel C: Plasma Hjv determination by a commercial ELISA kit. C: Control diet; ID: Iron-deficient diet; WT: Hjv+/+ mice; KO: Hjv−/− mice.

Fig 5: Hemojuvelin protein in mouse whole liver homogenates.C: Control, Bl: Bleeding (0.6 ml of blood once weekly for 3 weeks), Fe: Iron injection (200 mg/kg), KO: Hjv−/− mice. 80 µg of protein was loaded per lane. Arrows indicate Hjv-specific bands. Gapdh was used as loading control. Primary anti-Hjv antibody: AF3634.