FIGURE 5.

PP2A-Pep increased fast recycling of GABA_B receptors and reduced their sorting to lysosomal degradation. Cultured neurons were stressed or not (control) with glutamate (50 μM for 1 h) followed by incubation with PP2A-Pep. Neurons were then analyzed for the colocalization of GABA_B receptors with endosomal markers by in situ PLA using antibodies directed against GABA_B2 and EEA1 (A), Rab4 (B), and Rab7 (C). The location and shape of neurons analyzed in the representative images are outlined. Please note that PLA signals outside of the outlined neurons are associated with dendrites of adjacent neurons, which were not marked. (A) PP2A-Pep treatment increased the colocalization of GABA_B receptors with the early endosome marker EEA1. Left: representative images (scale bar, 10 μm). Right: quantification of PLA signals (white dots). The data represent the mean ± SD of 94–114 neurons per condition from five independent experiments. Two-way ANOVA followed by Tukey’s multiple comparison test (ns, p > 0.05; ****p < 0.0001). (B) PP2A-Pep treatment increased the colocalization of GABA_B receptors with the fast-recycling endosome marker Rab4. Left: representative images (scale bar, 10 μm). Right: quantification of PLA signals. The data represent the mean ± SD of 42–93 neurons per condition from three independent experiments. Two-way ANOVA followed by Tukey’s multiple comparison test (ns, p > 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001). (C) PP2A-Pep treatment reduced colocalization of GABA_B receptors with the late endosome marker Rab7. Left: representative images (scale bar, 10 μm). Right: quantification of PLA signals. The data represent as mean ± SD of 58–74 neurons per condition from three independent experiments. Two-way ANOVA followed by Tukey’s multiple comparison test (ns, p < 0.05, **p < 0.01, ****p < 0.0001).