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In vitro formation of ThiS thiocarboxylate using ThiF and ThiF(C184S). ThiF(C184S) or ThiF (74.9 μM) and ThiS-COOH (24.1 μM) were incubated together with ATP (5 mM), MgSO4 (2 mM), pyridoxal 5′-phosphate (50 μM), IscS (2 μM), and 35S-Cys (75 μM) and analyzed by Tris-tricine SDS/PAGE (39) followed by autoradiography using a PhosphoImager. RFI is a measurement of the relative fluorescence intensity.
