2.12. GABA transaminase enzymatic activity

T98G cells expressing non‐targeting control shRNA, ABAT shRNA, and ISCA2 shRNAs were harvested and GABA‐T enzymatic activity was determined using a GABA‐T assay kit (Biomedical Research Service Center, Buffalo University, NY) according to manufacturer instructions. Briefly, the assay is based on sequential GABA‐T transamination reaction and glutamate dehydrogenase reaction, which couples the reduction of iodonitrotetrazolium to iodonitrotetrazolium‐formazan (Ɛ = 18 mM⁻¹ cm⁻¹ at 492 nm). Each sample (10 μg) was assayed in duplicate in a 96‐well plate: one set for control wells (no substrate) and one set for reactions wells (containing GABA‐T substrate). After the incubation periods, the reactions were terminated by adding 3% acetic acid (Sigma–Aldrich, St. Louis, MO). The OD at 492 nm was measured using a plate reader (Phenix Sunrise series; Tecan). Readings were averaged and control wells readings were subtracted from sample wells readings (ΔOD). GABA‐T activity was calculated using the following formula: GABA‐T activity (μmol/(l min) = (ΔOD × 1,000 × 155 μl)/(60 min × 0.6 cm × Ɛ × 10 μl) = ΔOD × 23.92, where 155 μl is the total reaction volume, 0.6 cm the light path in the 96‐well plate, 10 μl the volume of sample in each well. The results were graphed as percentage of control.