Figure 1.

Nedd4-2 mediates the ubiquitination of glutamate transporter GLT-1 in MPP⁺-treated astrocytes. (a) Western blotting analysis showing that GLT-1 expression is decreased on MPP⁺ treatment of astrocytes for 24 h in the membrane. (b) l-[³H]-Glutamic acid uptake assay showing that 1 mM MPP⁺ and 2 μM PMA treatment for 24 h decreases the glutamate uptake in astrocytes. (c and d) Co-IP assays showing that GLT-1 is ubiquitinated after 24 h MPP⁺ treatment of astrocytes. The capture antibody in c was anti-GLT-1; and in (d) was anti-Ub. IgG was tested as a negative control. (e and f) Co-IP assay showing the interaction between Nedd4-2 and GLT-1 in MPP⁺ and PMA-treated astrocytes. The capture antibody in (e) was anti-Nedd4-2 and MPP⁺ treatment time was 24 h; the capture antibody in (f) was anti-GLT-1 and MPP⁺ treatment time was 24 h. IgG was tested as a negative control. (g) l-[³H]-Glutamic acid uptake assay showing that lysosome inhibitor (Leupeptin) while not proteasome inhibitor (MG-132) increased glutamate uptake in MPP⁺-treated astrocytes for 24 h. (h) Western blotting analysis showing that Leupeptin while not MG-132 increased GLT-1 expression in MPP⁺-treated astrocytes for 24 h. Results are expressed as the mean±S.E. of at least three experiments. Student's t-test for Figures 1a, c and d, and One-way ANOVA for Figures 1b, e, f, g and h. *Represents a significant difference between other group and control group. **P<0.01, *P<0.05. ^#Represents a significant difference between MPP⁺ group and Leupeptin+MPP⁺ group. ^#P<0.05