Fig. 5
Decreased cell surface levels of GluA1 and GluA2 receptors in VPS35-deficient neurons. a, b Reduced surface GFP-GluA1 in VPS35+/m hippocampal neurons. Both VPS35+/+ and VPS35+/m hippocampal neurons were transfected with GFP-GluA1 at DIV7. The surface GFP-GluA1 was labeled by GFP antibody (monoclonal) under non-permeabilizing conditions (no Triton X-100) at DIV 15, then the neurons were fixed. The total GFP-GluA1 were labeled by GFP antibody (polyclonal) after permeablized by Triton X-100. A, representative imunnostaining images. Scale bars, 5 μm. B, quantification of surface to total GFP-GluA1 in A. Data were shown as mean ± SEM; n = 30 neurons from 3 independent experiments; *p < 0.05. c, d Reduced surface GFP-GluA2 in VPS35+/m hippocampal neurons. As GFP-GluA1, GFP-GluA2 was transfected into VPS35+/+ and VPS35+/m hippocampal neurons at DIV7. The surface and total GFP-GluA2 were labeled by GFP antibody as described above. C, representative imunnostaining images. Scale bars, 5 μm. D, quantification of surface to total GFP-GluA2 in C. Data were shown as mean ± SEM; n = 30 neurons from 3 independent experiments; *p < 0.05. e, f Reduced cell surface level of GluA1 and GluA2 in VPS35+/m cortical neurons by biotinylation assay. E, representative blots; F, quantification of surface over total receptors (ratio) in E. Data were presented as mean ± SEM; n = 3; *p < 0.05