Figure 5.

EAAT2b surface expression in astrocytes. A, Streptavidin-Alexa 568 labeling (red) of EAAT2a-AP in primary astrocytes shows only a portion of the total protein (green) is at the cell surface. The robust labeling of EAAT2b-AP with the streptavidin-Alexa 568, however, demonstrates that it is found almost exclusively at the cell surface (bottom). Scale bars, 50 μm. B, Quantitation of the surface expression of these constructs by photon counting indicates that EAAT2b is found at the cell surface of primary astrocytes significantly more than EAAT2a. Addition of the PDZ ligand of EAAT2b to the EAAT2a construct can drive EAAT2a out to the cell surface. *p < 0.01; **p < 0.001 by one-way ANOVA (n = 10, 13, 16). Error bars indicate SEM. C, Immunoprecipitation of EAAT2 (both isoforms, bottom) from whole-brain lysates also isolates DLG1 (top), indicating binding of these two proteins. The PDZ domain-containing proteins PICK1 and PSD95 can be detected in total brain lysates, but not in the lysates from isolated EAAT2. D, Knockdown of DLG1 in astrocytes with shRNA decreases the membrane accessibility of EAAT2b-AP. ****p < 0.0001 by one-way ANOVA (n = 11, 10, 20). Error bars indicate SEM. E, Astrocytes in coculture with neurons (top) immunolabel for DLG1 (red) as well EAAT2 (green). Astrocytes in culture without neurons, however, have much lower signal intensity for the PDZ protein (bottom). F, Quantitation of membrane labeling of EAAT2a-AP or EAAT2b-AP in astrocytes in culture without neurons do not exhibit robust surface expression of either isoform (n = 4, 3). Error bars indicate SEM.