Supplementary Figure 5: Phosphorylated ephrin-B3 is localized outside synapses.
From: Anchoring and synaptic stability of PSD-95 is driven by ephrin-B3
(a) Phospho-S332 is enriched in the shaft of cultured cortical neurons. DIV21 neurons transfected with GFP at DIV0 were fixed and stained with the indicated antibodies at DIV21. Arrows indicate pS332 puncta in the dendritic shaft and arrowheads point to dendritic spines. Scale bar: 3µm.
(b) Quantification of pS332 labeling in different cellular compartments per unit area of GFP labeled dendrite or dendritic spine. Phospho-S332 puncta were significantly less abundant in spines compared to dendritic shafts. Data are represented as mean ± s.e.m., ANOVA, F (2, 42) = 5.98, Tukey’s post-hoc. *p = 0.0467, **p = 0.005, n = 15 neurons from 2 independent experiments.
(c) Source of western blot data for the PSD fractionation experiments shown in figure 3e. Probed as indicated at the bottom of each blot. Blots shown top to bottom in figure 3e are shown in left to right order. Boxes indicate approximate regions show in the main figure.
(d) Unphosphorylated ephrin-B3 is enriched in mouse cortical synaptosomes. P21 wild type mouse cortex fractionated into indicated fractions and labeled with the indicated antibodies. Purification of synaptosomes was confirmed by the enrichment of PSD-95 in these fractions along with ephrin-B3.
(e) Quantification of the pS332 signal relative to the total ephrin-B3 expression indicates that a significantly higher proportion of phosphorylated ephrin-B3 is found outside of synapses. Ratios of pS332 to ephrin-B3 were normalized to S1 fractions. Data are represented as the mean ± s.e.m., p<0.0001, ANOVA, F (2, 6) = 62.87, with Tukey’s post-hoc. **p = 0.0002, ***p = 0.0001, n = 3 wild type mice.
(f) Source of western blots shown in panel S5d. Probed as indicated at the bottom of each blot. Boxes indicate approximate regions show in the main figure.
(g) Source of western blots for the synaptosomes prepared from control and activity deprived cortex shown in figure 3i. Probed as indicated at the bottom of each blot. Blots shown top to bottom in figure 3i are shown left to right. Boxes indicate approximate regions show in the main figure.
(h) Source of western blots for the ephrin-B3 and PSD-95 synaptosome co-IP experiments prepared from control and activity deprived cortical hemispheres as shown in figure 3j. Probed as indicated at the bottom of each blot. Blots shown top to bottom in figure 3j are shown left to right. Boxes indicate approximate regions show in the main figure.
