Expression of DMT1 and iron uptake study. (A) Western blot analysis of peripheral blood cell lysates (90 μg) and Caco-2 cells lysate (30 μg) with primary goat anti-Nramp2 antibody (1/1000, 16 hours, 4°C) and peroxidase-conjugated secondary antibody (Pierce; 1/1000, 90 minutes, room temperature). The 64-kDa band represents the DMT1 protein; lane 1, Caco-2; lane 2, patient's father; lane 3, patient's mother; lane 4, patient. (B-C) Western blot analysis of total cell lysates (30 μg) and crude membrane extracts (50 μg) from CHO cells expressing the empty vector (CHO), WT, E399D, and DEL forms of HA-tagged DMT1 with mouse anti-HA antibody (1/1000, 1 hour, room temperature) and peroxidase-conjugated secondary antibody (1/1000, 90 minutes, room temperature). Mature complex-glycosylated DMT1 form (90 to 100 kDa; ▹) and the core-glycosylated form of DMT1 (66 kDa; ◂) are indicated. Equal loading of proteins was assessed by probing with an antibody against β-actin or α-tubulin (1/1000, 1 hour, room temperature). Representative immunoblots of 3 separate experiments are illustrated. (D) Iron transport activities of WT, E399D, and DEL DMT1 incubated in pH 6.0 incubation buffer with ⁵⁹Fe(II)-ascorbate (10 μM ⁵⁹Fe). Iron uptake is expressed as intracellular ⁵⁹Fe (cpm per 10⁶ cells). “CHO empty” represents iron uptake by cells transfected with empty vector. Data shown are the means ± SD of duplicate determinations from a typical experiment that was performed 3 times.