Supplementary Figure 4: Activity-dependent regulation of Ser332 phosphorylation underlies ephrin-B3 localization.
From: Anchoring and synaptic stability of PSD-95 is driven by ephrin-B3
(a) Source of the western blot data shown in figure 3b. Boxes indicate approximate regions show in the main figure. Probed as indicated at the bottom of each blot.
(b) Treatment of neurons with PD98059 blocks ephrin-B3 S332 phosphorylation. Western blots of neuronal lysates from DIV14 neurons treated with TTX (1 µM, 4 hours, Tocris) and MEK1/2 inhibitor PD98059 (100 µM, 4 hours, Sigma) as shown and probed with the indicated antibodies. 55 mM KCl (1 hour) was used to depolarize the neurons and induce Erk activation.
(c) Quantification of pS332 signal relative to total ephrin-B3. Depolarization induces a significant increase in pS332 signal that was blocked by pretreating neurons with PD98059. Data are shown as mean ± s.e.m., p = 0.0205, ANOVA, F (3, 8) = 5.843, with Fischer’s LSD post-hoc. *p = 0.0154 (TTX - KCl), **p = 0.0041 (TTX-PD - KCl), *p = 0.0298 (KCl - KCl+PD), n = 3 independent experiments.
(d) Source of the western blot data shown in panel S4b. Boxes indicate approximate regions show in the main figure. Probed as indicated at the bottom of each blot.
