Figure 3.

Apparent affinity of second phase Fe²⁺ uptake in mitochondria from rDMT1-overexpressing HEK293 cells is consistent with DMT1-mediated transport. PGSK-loaded mitochondria (pH 7.6_i) from doxycycline-induced rDMT1-HEK293 cells were assayed for quenching induced by increasing Fe²⁺ concentrations. Fe²⁺ was added from 500x concentrated FeSO₄ stock solutions, which had been supplemented with 50% cytosol, as detailed in Materials and Methods. The initial 20s-slopes of second-phase PGSK quenching (−ΔPGSK), as described in Fig. 1, were normalized to values obtained at the maximally tested concentration of 10 µM Fe²⁺. Data represent means ± SEM of 4–7 independent experiments. Data were fitted to a one-site saturation binding function, yielding a half-saturation concentration $(K_{0.5}^{F{e}^{2+}})$ of 1.3 µM Fe²⁺. RFU, relative fluorescence units.