Supplementary Figure 10: D-___domain and Ser332 of ephrin-B3 are required for normal localization of PSD-95.
From: Anchoring and synaptic stability of PSD-95 is driven by ephrin-B3
(a) Effects of ephrin-B3 shRNA and molecular replacement of ephrin-B3 on the distribution of PSD-95-GFP in cultured cortical neurons. Representative images of PSD-95-GFP (arrows) in dendritic segments from neurons transfected with PSD-95-GFP, ephrin-B3 shRNA, and the indicated flag-tagged ephrin-B3 mutant rescue constructs. Plot profiles under the images indicate dendritic distribution of GFP fluorescence in each of the transfection conditions. The dashed line represents the intensity of GFP in dendritic shafts of control neurons (Figure 4h). Flag-ephrin-B3-L293A and phosphomimetic flag-ephrin-B3-S332D, which exhibit reduced interaction with PSD-95, do not rescue diffuse dendritic PSD-95-GFP distribution caused by the knockdown of endogenous ephrin-B3 (Figure 4 in the main text). Rescuing ephrin-B3 expression with non-phosphorylatable flag-ephrin-B-3S332A restores punctate PSD-95-GFP localization (arrows) and reduces diffuse dendritic GFP fluorescence to control levels (dotted line in profile plots). Scale bars: 3 µm.
(b) Quantification of GFP intensities per pixel in dendritic shafts. D-___domain flag-ephrin-B3 (n = 10 neurons) and phosphomimetic flag-ephrin-B3_S332D (n = 10 neurons) rescue conditions exhibit higher shaft GFP fluorescence than control neurons. Non-phosphorylatable ephrin-B3-S332A mutant (n = 8 neurons) rescues shaft GFP fluorescence back to control levels. p<0.0001, F (4, 57) = 7.233, ANOVA with Tukey’s post-hoc. Control (n = 17 neurons), eB3 shRNA (n = 17 neurons) – p = 0.0074; Control, L293A (n = 10 neurons) – p = 0.0307; Control, S332D (n = 10 neurons) – p = 0.0015; Ephrin-B3 shRNA, S332A (n = 8 neurons) – p = 0.0208; L293A, S332A – p = 0.0478; S332A, S332D – p = 0.0042.
(c) Quantification of GFP intensities per pixel in puncta. ANOVA, F (4, 57) = 10.58, p < 0.0001, Tukey’s post-hoc. Control (n = 17 neurons), eB3 shRNA (n = 17 neurons) – p = 0.0003; Control, L293A (n = 10 neurons) – p = 0.0146; Control, S332D (n = 10 neurons) – p = 0.0018; Ephrin-B3 shRNA, S332A (n = 8 neurons) – p = 0.0001; L293A, S332A – p = 0.0033; S332A, S332D – p = 0.0005.
