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After the washing step, the TMB substrate solution was added to each well followed by incubation at 37 C for 20 min

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The Intracellular ferrous iron can be oxidized and exported by the membrane protein ferroportin (FPN), which currently is the unique iron efflux pump discovered that can release iron from cells ( The intracellular labile iron levels could be enhanced via TF-mediated iron absorption or selective autophagy of ferritin