Time and temperature dependence of Mn²⁺ and Fe²⁺ transport by Nramp1HA and Nramp2HA measured by a radioisotopic uptake assay. Incorporation of ⁵⁵Fe²⁺ (A) and ⁵⁴Mn²⁺ (B) into CHO control cells, and Nramp1HA expressing (clone N1-94) or Nramp2 HA expressing (clone N2-310a) CHO cell transfectants was measured as described in “Materials and methods.” Briefly, cells were resuspended in transport buffer (10⁷ cells/1.5 mL), and transport was initiated by addition of 1 mL of radioisotope buffer containing tracer amounts of either ⁵⁴Mn²⁺ (total Mn²⁺ concentration of 9 μM) or ⁵⁵Fe²⁺ (total Fe²⁺ concentration of 9 μM), followed by a 30-minute incubation at 20°C. At predetermined time points (0, 5, 15, 30 minutes), metal accumulation was calculated and expressed as picomolar equivalents (pmoleq) divalent-metal/μg total cellular protein as shown in panels A (Fe²⁺) and B (Mn²⁺), which represent the average (with standard error) from 3 or 4 independent experiments. Parallel experiments were conducted at 4°C to establish the temperature-independent component of cell-associated radioactivity (binding). These values were subtracted from the 20°C accumulation data to deduce net uptake values, which are shown in panels C (Fe²⁺) and D (Mn²⁺).