Detection of DMT1 protein in duodenum. Total duodenal extract was prepared as described: 22 μg total protein was loaded into each lane and separated by SDS-PAGE on a 10% acrylamide gel followed by transfer to PVDF membrane. Membranes were cut in half just below the 50-kDa standard; the upper half of the blot was incubated with the nRAMP2-NT antibody, whereas the lower half was incubated with the GAPDH antibody as an internal loading control.