Ferroportin downregulation is mediated by TLR2 and TLR4 ligands whereas hepcidin activation is limited to the TLR4 ligand LPS in BMDMs. (A,C-D) Ferroportin mRNA expression was determined by qRT-PCR in WT (A), TLR6-deficient (C), and TLR2-deficient (D) BMDMs stimulated with 100 ng/mL TLR2 ligands (FSL1, PAM3CSK4, PamOct2C-(VPG)4VPGKG) or TLR4 ligand (LPS) for the indicated time. (B) Western blot analysis and quantification of ferroportin expression in BMDMs from WT mice treated with 100 ng/mL LPS and PAM3CSK4 for 24 hours. β-actin was used as loading control. (E-G) Hepcidin mRNA expression was analyzed in the same samples. The mRNA quantification was calibrated to 36B4 mRNA levels. All data are reported as means ± SEM; BMDMs were derived from at least 4 different mice per group. Each lane in the Western blot analysis represents the protein lysate obtained from a single mouse. *P < .05; **P < .01; ***P < .001; ****P < .0001; Student t test.