FIGURE 3.

Extracellular microvesicles from astrocytes contain functional EAAT-1. (A) Representative western blots showing NaK ATPase, EAAT-1 and glutamine synthetase in astrocyte-derived eMV, whereas only NaK ATPase is detected in eMV from microglia or serum. Lanes correspond to medium from 75 cm² of confluent cells. Coomassie staining (coom) shows total protein content. (B) Western blots of astrocyte (GFAP positive) and microglia (Iba-1 positive) extracts showing that both cell types express EAAT-1 in culture (10 μg of proteins extracts loaded). (C) Bar graph showing [³H] aspartate reuptake from astrocyte-conditioned medium treated (black bar) or not (white bar) with the specific EAAT blocker TF-TBOA. Given as mean ± SEM. **p < 0.001, t-test with Welch correction, n = 3 samples from 3 independent dishes. (D) Time course study of eMV composition in response to PMA without medium replacement. An enrichment of NaK ATPase and glutamine synthetase is found at 6 and 9 h post-treatment together with an increase of EAAT-1 signal after 9 h, but without modifications of Coomassie staining. Bar graph shows quantification of EAAT-1. **p < 0.01, one way ANOVA followed by Dunnett’s post hoc test vs. control column (n = 4 samples from independent cultures for each condition). (E) Time course study (30 min and 6 h) of microvesicular NaK ATPase and EAAT-1 in response to PMA after a full medium change. A significant augmentation of EAAT-1 is observed 6 h post-PMA in comparison to eMV from vehicle treated cells.*p < 0.05, one way ANOVA followed by Dunnett’s post hoc test vs. control column (n = 4 samples from independent cultures). (F) Western-blots showing the PKC isoforms activated in primary astrocytes after PMA treatment. PMA treatment increases phospho-PKC-δ and -θ. Coomassie staining (coom) shows the total protein content. (G) Western blot showing that rottlerin counteracts the increase of EAAT-1 in eMV after PMA. The bar histogram shows the quantification of band intensities. *p < 0.05, one way ANOVA followed by Dunnett’s post hoc test vs. control column (n = 3 independent cultures for each condition).