Functional consequences of the human DMT1 (SLC11A2) mutation on protein expression and iron uptake by Monika Priwitzerova, Guangjun Nie, Alex D. Sheftel, Dagmar Pospisilova, Vladimir Divoky, and Prem Ponka Blood Volume 106(12):3985-3987 December 1, 2005 ©2005 by American Society of Hematology

Expression of DMT1 and iron uptake study. 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. Monika Priwitzerova et al. Blood 2005;106:3985-3987 ©2005 by American Society of Hematology

Immunofluorescence analysis of DMT1 in BFU-E-derived erythroblasts and transfected CHO cells. Immunofluorescence analysis of DMT1 in BFU-E-derived erythroblasts and transfected CHO cells. (A) Day 14 healthy control and the patient's BFU-Es were harvested, cytospun, and stained using goat anti-Nramp2 antibody (1/50, 3 hours, room temperature) and fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Molecular Probes, Eugene, OR; 1/1000, 90 minutes, room temperature). (B) Immunostaining of WT (i-ii), E399D (iii-iv) and DEL (v-vi) DMT1 in transiently transfected CHO cells with mouse anti-HA antibody (1/200, 1 hour, room temperature) and FITC-conjugated secondary antibody (1/1000, 1 hour, room temperature). (C) Subcellular localization of E399D DMT1 in early endosomes (i-iii) and DEL DMT1 in the endoplasmic reticulum (iv-vi) in stably transfected CHO cells. Cells immunostained with mouse anti-HA antibody and with FITC-conjugated secondary antibody were subsequently stained either with anti-EEA1 antibody (1/200, 1.5 hours, room temperature) or anti-calnexin antibody (1/200, 1.5 hours, room temperature) followed by incubation with red-fluorescent-conjugated secondary antibody (Alexa Fluor 594, Molecular Probes; 1/1000, 1.5 hours, room temperature). In panels A and C the cells were visualized on an Olympus BX 50 fluorescence microscope (Olympus, Hamburg, Germany) using a 100 ×/1.3 numeric aperture (NA) oil immersion objective. Digital images were acquired with an Olympus DP 50 camera driven by the software Viewfinder Lite version 1.0.135 (Pixera, Los Gatos, CA). Original magnifications, × 1000. In panel B the cells were examined on a Zeiss Pascal 5 confocal microscope (Carl Zeiss, Jena, Germany) using a 40 ×/0.75 NA objective (i, iii, v) and a 63 ×/1.4 NA oil immersion objective (ii, iv, vi). The Zeiss LSM Browser version 3.2.0 115 was used for handling pictures. Images were cropped, assembled, and labeled using Adobe Photoshop software (Adobe Systems, San Jose, CA). Monika Priwitzerova et al. Blood 2005;106:3985-3987 ©2005 by American Society of Hematology