GLT-1 binds to Hsp90β, and 17AAG prevents GLT-1–Hsp90β–20S proteasome assembly. (A) Representative Western blot of astrocytes treated with DMSO or 17AAG (100 nM) in the presence of CHX (50 µg/ml) before harvesting (left). (right) Quantification of band intensities (normalized to actin and the t = 0 time point; n = 3). (B) Representative Western blot of astrocytes treated with 17AAG (100 nM), CHX (50 µg/ml), MG132 (10 µM), or both for 24 h, as indicated (left). Statistical analysis of GLT-1 expression (right; *, P < 0.05, Student's t test). (C) Representative Western blot of the pulse–chase analysis. Astrocytes were preincubated with sulfo-NHS-SS-biotin for 30 min to label surface proteins and were then treated with DMSO or 17AAG (400 nM; n = 3; *, P < 0.05, Student's t test). Equal protein loading was confirmed by silver staining. (D) Representative Western blot analysis for GLT-1-FLAG after the immunoprecipitation of Hsp90β-myc. 293t cells were cotransfected with Hsp90β-myc and GLT-1-FLAG for 48 h and lysed for immunoprecipitation with an anti-myc antibody. (E) Representative Western blot analysis for Hsp90β-myc after immunoprecipitation of GLT-1-FLAG (n = 3). 293t cells were cotransfected with Hsp90β-myc and GLT-1-FLAG. 6 h after transfection, the cells were treated with or without 17AAG for 42 h (right; normalized to actin; **, P < 0.01, Student's t test). (F) PLA assay for GLT-1-Hsp90β association in astrocytes treated with 17AAG at different concentrations for 48 h. The nuclei were stained with DAPI (blue). The data are expressed as the median, the 25th–75th percentile (box), or the min-max percentile (whiskers; n = 3; *, P < 0.05; ***, P < 0.001, one-way ANOVA, Tukey's post hoc test). Bar, 10 µm. Data are representative of at least two independent experiments.