Ggt colorimetric assay kit

The γ-glutamyltransferase (GGT) activity was assessed using the GGT colorimetric assay kit (Sigma-Aldrich, MAK089). The kit is based on the enzymatical cleavage of L-γ-Glutamyl-p-nitroanilide (γ-Glu-pNa) by GGT liberating the chromogen p-nitroanilide, whose formation can be monitored at 418 nm. To assess the effect of GS-SO3 on the GGT activity, increasing concentrations of GS-SO3 (between 130 μM and 2.5 mM) were added to the reaction and incubated for up to 1 h at 37°C with constant monitoring of the absorbance. Upon 1 h, the reaction was stopped by incubating on ice for 5 min and the mixture was measured using LC-MS and the method described in 2.5.2. Data analysis was performed using Progenesis QI (Non-linear Dynamics/Waters, Newcastle upon Tyne, United Kingdom).

The activity assay was performed using the GGT colorimetric assay kit (Sigma Aldrich; St. Louis, MI, USA) per the manufacturer’s instructions. Tissue samples were prepared using approximately 10 mg of kidney cortex in 200 μL of ice-cold GGT assay buffer. The samples were then centrifuged at 13,000× g for 10 min to remove insoluble material. The supernatant was kept and further diluted to 1:100 using the GGT assay buffer and stored at −80 °C until further use. 4 h: (WT vehicle: n = 6), (NHERF1 KO vehicle: n = 6), (WT cisplatin: n = 6), and (NHERF1 KO cisplatin: n = 6) and 24 h: (WT vehicle: n = 5), (NHERF1 KO vehicle: n = 5), (WT cisplatin: n = 7), and (NHERF1 KO cisplatin: n = 8).