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).