FSL1-mediated TLR2/6 ligation reduces ferroportin expression in BMDMs without activating hepcidin mRNA expression. (A,C) qRT-PCR analysis of ferroportin mRNA in BMDMs from WT and TLR6-deficient mice (A), and from WT and TLR2-deficient mice (C) stimulated with FSL1 (20 ng/mL or 100 ng/mL) for the indicated time. (B,D) Western blot analysis and quantification of ferroportin expression in BMDMs from WT and TLR6-deficient mice (B) and from WT and TLR2-deficient mice (D) treated with 100 ng/mL FSL1 for 24 hours. β-actin detection ascertains equal sample loading. (E-F) Ferroportin and hepcidin mRNA expression in BMDMs after FSL1 and LPS (100 ng/mL) stimulation. mRNA levels were normalized 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; Student t test.