The decrease in splenic ferroportin levels is independent of hepcidin activation. WT mice were injected with saline (ctrl), FSL1, or LPS (25 ng of ligand per gram of body weight) and the spleens were analyzed 3 hours after injection. (A-D) Ferroportin, hepcidin, TNFα, and IL6 mRNA expression was assessed by qRT-PCR. (E-G) Western blot analysis and quantification of ferroportin expression in the whole spleen (E-F) and in splenic macrophages isolated from the injected mice (G). β-actin was used as loading control. (H) Splenic non-heme iron content and DAB-enhanced Perls’ iron staining show iron retention in red pulp macrophages of the spleen of FSL1- and LPS-injected mice. Each lane in the western blot analysis represents the protein lysate obtained from a single mouse. Data are means ± SEM. *P < .05; **P < .01; ***P < .001; Student t test; n = 6 mice per group.