Fig. 10
P150Glued deficiency increases protein expression of glutamate receptors and vulnerability to glutamate-mediated excitotoxicity in spinal neurons. a Western blot analysis shows the expression of AMPAR subunits GLUR1 and GLUR2, NMDAR subunits NR2A and NR2B, postsynaptic density protein 95 (PSD95) and synaptophysin (SYP) in the spinal cord homogenate of 9-month-old Dctn1LoxP/LoxP and Dctn1LoxP/LoxP; Thy1-Cre mice. Actin was used as loading control. b Bar graphs show the quantification of GLUR1, GLUR2, NR2A, NR2B, PSD95 and SYP level normalized with actin (n = 10 per genotype). Data were presented as mean ± SEM; **p < 0.01, ***p < 0.001. Unpaired t-test was used for statistical analysis. c Western blot analysis shows the levels of biotinylated cell surface and total AMPAR subunits GLUR1 and GLUR2, NMDAR subunits NR2A and NR2B in cultured Dctn1LoxP/LoxP and Dctn1LoxP/LoxP; Cre/Esr1 spinal neurons at 21 DIV. Actin was used as loading control. d Bar graphs show the quantification of total and surface GLUR1, GLUR2, NR2A and NR2B level normalized with actin, and the ratio of GLUR1, GLUR2, NR2A and NR2B at cell surface versus total proteins (n = 8 per genotype). Data were presented as mean ± SEM; **p < 0.01, ***p < 0.001. Unpaired t-test was used for statistical analysis. (E) Primary spinal neurons were treated with glutamate, and the viability of these neurons was measured by the MTT assay. Box & whiskers graphs show survival rate (percentage) of cultured Dctn1LoxP/LoxP and Dctn1LoxP/LoxP; Cre/Esr1 spinal neurons (21 DIV) treated with 0 or 10 μM glutamate for 24 h (N = 40 per genotype per condition). Data were presented as mean ± SEM. One-way ANOVA plus Tukey’s post hoc test was used for statistical analysis. ***p < 0.001 for difference between Dctn1LoxP/LoxP neurons treated with 0 and 10 μM glutamate, ***p < 0.001 for difference between Dctn1LoxP/LoxP; Cre/Esr1 neurons treated with 0 and 10 μM glutamate, **p < 0.01 for difference between Dctn1LoxP/LoxP and Dctn1LoxP/LoxP; Cre/Esr1 neurons treated with 10 μM glutamate, while no difference was found between Dctn1LoxP/LoxP and Dctn1LoxP/LoxP; Cre/Esr1 neurons treated with 0 μM glutamate