Knockdown of GMFG in macrophages alters iron metabolism protein expression to mimic that observed in the M2 macrophage phenotype. (A) Immunoblot analysis of iron metabolism proteins in cellular lysates of RAW264.7 macrophages transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 (LPS/IFN-γ) or M2 (IL-4/IL-13) macrophage inducers for 24 hours. α-tubulin was used as a loading control. (B) Immunoblot analysis of IRP1, IRP2, and HIF-2α in cellular lysates of RAW264.7 macrophages transfected with control siRNA or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 or M2 macrophage inducers. α-tubulin was used as a loading control. (C) Representative RNA-binding activity of IRP1 analyzed by electrophoretic mobility shift assays. RAW264.7 macrophages were transfected with control siRNA or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 or M2 macrophage inducers for 24 hours. Cytoplasmic lysates were incubated with an excess of a phosphorus-32–labeled iron regulatory element probe. RNA-protein complexes were resolved on nondenaturing polyacrylamide gels and revealed by autoradiography.