C2978

Whole cell lysate was prepared with lysis buffer (C2978, Sigma-Aldrich). Three micrograms of PC3 cell whole lysate was used for pyruvate kinase assay (ab83432, abcam) following manufacturer’s instruction. Kinetic measurement at O.D 570 nm by microplate reader (Hidex Sense Beta Plus) recorded pyruvate kinase activity every minute for 40 min.

Cultured astrocytes in 24-well plates were washed twice and then incubated in Krebs buffer (135 mM NaCl, 5 mM KCl, 0.6 mM MgSO₄, 1 mM CaCl₂, 6 mM d-glucose, and 10 mM HEPES; pH 7.5) containing 20 nM ³H-glutamate (0.5 μCi; specific activity = 50.8 Ci/mmol; PerkinElmer) and 40 μM unlabeled glutamate for 10 minutes at 37°C. DHK was used to distinguish DHK-sensitive (EAAT2) ³H-glutamate uptake from DHK-insensitive uptake. After incubation, the Krebs buffer was replaced with PBS to stop the uptake of glutamate, and the cells were then collected in cell lysis solution (C2978; Sigma-Aldrich). The lysates were processed to measure both ³H-glutamate levels with a liquid scintillation counter (LS6500 scintillation counter; Beckman Coulter) and protein content by using the Bradford Protein Assay kit (5000002; Bio-Rad). The concentration of glutamate taken up by the astrocytes (in nanomolars) was calculated using the radioactivity and the total protein concentration per milligram of protein over a 10-minute period. DHK-sensitive ³H-glutamate uptake was calculated as the total amount of Na⁺ dependent ³H-glutamate taken up minus the amount of DHK-insensitive Na⁺-dependent ³H-glutamate taken up (DHK-treated samples). All values were normalized to mean glutamate uptake in the control group, and all measurements were repeated 3 times.