Cells were collected in lysis buffer (100 mM Tris pH 7.6, 5 mM EDTA, 1 mM NaN3 and 0.1% peroxide-free Triton-X100). Lysates were complemented with 0.6 U/mL glutathione reductase, 0.2 mM nicotinamide adenine dinucleotide phosphate hydrogen, 3 mM reduced glutathione and 200 μM of the substrate cumene hydroperoxide. NADPH turnover was measured on an BioTek Synergy reader at 340 nm over 10 min at 37° C. Enzymatic activity was calculated after subtracting absorbance decay obtained from buffer without cell lysates by using NADPH extinction and by normalizing to total protein content [40 (link)].
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