Fig 2: The expression level of a group of synaptic proteins is not altered in MAP1B KO neurons.Immunoblots against the presynaptic proteins Bassoon, Synaptotagmin I (Sytg I), Synaptophysin I (Synph I) and SNAP-25, and against the postsynaptic proteins Homer 1 and PSD-95. Expression of MAP1B in the different genotypes is shown, and tubulin was used as a loading control. 30 μg of protein were loaded in each lane. (A) Proteins derived from E18.5 embryonic brains of WT, MAP1B heterozygous and MAP1B KO mice. (B) Proteins from mature cortical neurons derived from WT and MAP1B KO mice. One–way ANOVA with Bonferroni post-test showed no significant differences between conditions, n = 3 for each condition (A). Unpaired Student’s t-test showed no significant differences between conditions, n = 3 for each condition (B). For (A,B) gels for each antibody were run under same experimental conditions and blot were processed in parallel. Western blot raw data is presented in Supplementary information.

Fig 3: PSD-95 expression pattern depends on culture duration. (A, B) Representative tdTomato-positive hRGCs (red) cultured for 1 (A) and 4 (B) weeks and immunolabeled against PSD-95 (green). Scale bar = 20 µm. (C) PSD-95 labeling within hRGC somas significantly decreased after 4 weeks versus earlier time points (P ≤ 0.033). (D) Quantification of PSD-95 labeling within hRGC primary neurites. PSD-95 labeling significantly increased by 3 and 4 weeks in culture compared with 1-week cells. Significance indicators: * comparison with 1-week cells, # comparison with 2-week cells, ^ comparison with 3-week cells. Statistics: one-way analysis of variance, Tukey post hoc test (C, D). Data are mean ± standard error of the mean.

Fig 4: Short-term NMDAR overactivation induces its internalization from the synaptosome. Representative Western blot and densitometric analysis of NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM proteins in the synaptosomal fraction that was isolated from primary neurons in vitro. PSD95 and calnexin were used as synaptosome and ER markers, respectively. Total protein levels, determined by Ponceau S staining (see "Materials and methods" section), were used as loading controls. The data are expressed as the mean ± SEM of four-to-six independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired t-test)