The lack of Bmp6 and/or Hjv does not prevent the induction of hepcidin by LPS but the level of hepcidin reached after stimulation is linked to baseline BMP/Smad signaling in unchallenged animals. (A) Hepcidin (Hamp) mRNA expression was measured by quantitative PCR before and after LPS stimulation in the liver of male (M) and female (F) littermates that had the WT, Bmp6^−/−, Hjv^−/−, or double knockout (KO) genotype. (B) Serum hepcidin levels were measured by competitive ELISA on the same mice. Means ± standard deviation (SD) are shown on the graphs. Data on LPS-challenged mice were obtained on 8 WT, 4 Bmp6^−/−, 9 Hjv^−/−, and 5 double knockout males, and on 6 WT, 4 Bmp6^−/−, 7 Hjv^−/−, and 4 double knockout females. Means of quantitative PCR ΔCt values and log-transformed serum hepcidin before and after LPS were compared with Student t tests. (C) Representative western blot of 7 independent experiments for phospho-Smad5, total Smad5, phospho-Stat3, and total Stat3 expression in the liver of WT (Bmp6⁺/Hjv⁺), Bmp6^−/−(Bmp6⁻/Hjv⁺), Hjv^−/− (Bmp6⁺/Hjv⁻), and double knockout (Bmp6⁻/Hjv⁻) male littermates before (LPS⁻) and after (LPS⁺) LPS stimulation. Western blots were analyzed on a Chemidoc MP Imaging System (Bio-Rad). (D) Quantification was obtained with Image Laboratory Software. Means of PSmad5-to-Smad5 ratios were compared with 2-way ANOVA. Results of comparisons of unchallenged knockout with unchallenged WT mice as well as of LPS-challenged with unchallenged mice of the same genotype are shown above the bars. Results of comparisons between knockout mice are shown by connecting lines. *P < .05; **P < .01; ***P < .001; ****P < .0001.