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Fig. 6. KA stimulation promotes the degradation of internalized EAAC1 in the lysosome. (A,B) Double-immunofluorescence labeling showing that internalized EAAC1 is colocalized with the early endosomal marker EEA1 (A) and the lysosomal probe LysoTracker Red DND-99 (B) in C6 glioma cells upon a 30-minute stimulation with 10 µM KA. Bars, 10 µm. Scatterplot graphs of the individual pixels from the paired images and the Pearson's correlations calculated by Image-Pro-Plus software, suggesting that the colocalization of EAAC1 with EEA1 or Lysotracker Red DND-99 is significantly enhanced after KA stimulation. (C) Representative immunoblot and quantitative analysis show that incubation with 10 µM KA for 6 hours leads to the degradation of the internalized EAAC1, which is blocked by the lysosomal degradation inhibitor leupeptin (100 µM) or the compartment acidification blockers NH₄Cl (50 mM) and chloroquine (200 µM). ^**P<0.01 compared with the cells without KA stimulation (n=4). (D) Representative immunoblot and quantitative analysis showing that upon 10 µM KA stimulation the degradation of the internalized EAAC1 increases in a time-dependent manner. Additionally, Syn1A siRNA blocks the KA-promoted EAAC1 degradation. ^*P<0.05, ^**P<0.01 compared with the cells transfected with NS siRNA (n=4).

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