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Figure S1. Effect of the VRAC blocker DCPIB on intracellular content of L-glutamate, Lglutamine, taurine, and L-alanine. Primary rat astrocytes were incubated in Basal media for 30 min with or without 20 μM DCPIB. Levels of 5 intracellular amino acids were analyzed in cell lysates using an HPLC assay and normalized to protein content. Data are the mean values ± SE of 6 experiments. ***p<0.001, vs. basal. Inset shows no changes in glutamine levels in DCPIB-treated cells on an expanded scale.

Figure S2. Effect of the VRAC blocker DCPIB on enzymatic activities of (a) glutamine synthetase and (b) glutaminase. Primary rat astrocytes were incubated in Basal media with L-[3H]glutamate or L- [3H]glutamine in the presence or absence of 20 μM DCPIB. Enzymatic activities were quantifies by measuring conversion of L-[3H]glutamate to L-[3H]glutamine for glutamine synthetase (GS), or L-[3H]glutamine to L-[3H]glutamate for glutaminase (GLNase) as described in Methods. In each set of experiments enzymatic activities were normalized to controls to account for culture-to-culture variability. Data are the mean values ± SE of 6 experiments for GS (a) and 3 experiments for GLNase (b). *p<0.05 vs. 100%, t-test.

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