Ndfip1^−/− mice show increased levels of DMT1 in the liver associated with iron loading. (A) Immunoblot analysis of DMT1 in liver tissue of 6-week-old Ndfip1^+/+ and Ndfip1^−/− mice. There is an increase in the levels of DMT1 in Ndfip1^−/− mice compared with Ndfip1^+/+ mice; 10% input was probed for β-actin as loading control. (B) DMT1 protein levels were quantitated relative to the β-actin loading control and displayed graphically. (C) Perl's iron staining of paraffin-embedded liver sections. Iron (stained in black) is seen accumulating around the portal veins in the Ndfip1^−/− liver. The sections were counterstained with nuclear fast red to delineate cell nuclei. Data from 3 different Ndfip1^−/− animals and their wild-type Ndfip1^+/+ littermates are shown. (D) Transport activity in primary hepatocytes is increased in Ndfip1^−/− mice compared with wild-type littermates. The relative transport activity was measured by fluorescence quenching assay as described in Figure 1. *P < .05. Data are mean plus or minus SEM. Images were viewed with an Olympus BX51 microscope (Olympus, Center Valley, PA) using a UplanApo Lens at 40×/0.85 NA. Images were acquired using an Olympus camera model DP70 (3.0) and processed with Olysia Bioreport version 3.2 (Olympus) software. Images were compiled using Adobe Photoshop version 6.0 software (Adobe Systems, San Jose, CA).