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To demonstrate the single covalent modification by RSL3 is not on cys46, we expressed and purified tag-free GPX4 allCys(−) A46C protein, in which A46 in allCys(−) GPX4 was reverted to C46 (Figure 2A)

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To exert that activity, 5-FU requires intracellular conversion into cytotoxic metabolites (i.e., the body's cells convert it into active molecules that kill cancer cells)