17AAG enhances the glutamate clearance capacity of astrocytes in vitro. (A) Representative Western blot analysis for membrane and cytosolic GLT-1 (left). Astrocytes were treated with 17AAG (100 nM, 48 h). The bar graph shows the relative fold change of GLT-1 (right; n = 3; *, P < 0.05; **, P < 0.01, Student’s t test). (B–D) [³H]-glutamate uptake analysis. Astrocytes were pretreated with 17AAG (B; 100 nM, 48 h; n = 4), Hsp90-siRNA (C; 40 nM, 48 h; n = 5), or Hsp90β lentivirus (D; 72 h; n = 5), and DHK (100 µM), a GLT-1 inhibitor, was added 1 h before the assays to distinguish DHK-sensitive glutamate uptake (**, P < 0.01; ***, P < 0.001, Student’s t test). (E and F) Extracellular glutamate assay. 36 h after DMSO or 17AAG (100 nM) treatment, the astrocytes were depleted of glutamate for 12 h in Opti-MEM, and then incubated in an uptake buffer containing 500 nM or 20 µM unlabeled glutamate for (E) short-term or (F) long-term assays, respectively. Extracellular glutamate concentrations were determined in samples from the uptake buffer collected at the indicated time points (n = 4 for each group; *, P < 0.05, Student’s t test in E; *, P < 0.05; **, P < 0.01, one-way ANOVA with post-hoc Dunnett's tests in F). All experimental data were verified in at least two independent experiments.