Iron excess increases the phosphorylation and membrane localization of PKCα. (A) WT mice, after 4-hour starvation, received 50 mg/kg FeSO₄ via gavage. After the indicated times, mice were euthanized, and villi were isolated for western blotting analysis of the phosphorylation of PKCα (pPKCα) with anti–phosphorylated-PKCα (Thr638) antibody. (B) BMDMs were treated with ferric ammonium citrate (FAC, 200 μM) for the indicated times, and the expression of pPKCα was determined. (C) BMDMs were treated with FAC, and the localization of PKCα was detected by staining with anti-PKCα antibody and then confocal imaging. White arrow, plasma membrane expression of PKCα. WT mice were treated with and without mouse hepcidin (20 μg per mouse) for 24 hours. (D) Labile Fe²⁺ content was determined in whole-mount duodenal tissue and the cross sections by staining with FerroOrange. White arrowhead, intestinal villous tip. The expression of pPKCα was analyzed in the duodenum and spleen by western blotting (E). Data are expressed as mean ± SEM of 3 independent experiments for panel B. ∗P < .01; and ∗∗P < .05 compared with the untreated controls. Bar, 10 μm for panel C or 100 μm for panel D.