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We further investigated whether Ggt1-induced glutamate and its products perturbed downstream pathways

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For reducing SDS-PAGE, 2 g of protein was mixed with 3 L of 4 Bolt LDS Sample Buffer (Invitrogen), 1.2 L of 10 Bolt Sample Reducing Agent (Invitrogen), and Milli-Q water to a total volume of 12 L, and the samples were incubated for 10 min at 95 C

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After mixing the sample or standard with the detection reagent and incubating at room temperature for 15 minutes, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 390 nm