Figure 4. EphrinB2 interacts with VEGFR2 in hippocampal neurons.

(A) Cell lysate from primary wild type hippocampal neurons 14 DIV was used for co-immunoprecipitation. To pulldown ephrinB2, the beads were coupled with EphB4 receptor ectodomain fused to Fc (EphB4-Fc). Fc fragment served as control. Western blots performed using anti-VEGFR2 and anti-ephrinB2 antibodies show interaction of ephrinB2 and VEGFR2. (B) Wild type primary hippocampal neuron cultures were cultured for 14 DIV transfected with EGFP and proximity ligation assay (PLA) was performed using EphB4-Fc and an anti-VEGFR2 specific antibody. Magenta puncta represent amplified PLA signal and reflect the interaction between VEGFR2 and ephrinB2. PLA puncta localize to dendritic spine heads and necks labeled with EGFP (green) (indicated by arrowheads). Scale bar: 10 µm. (C) Labeling of hippocampal neuron cultures at 14 DIV with antibodies against PSD95 (green) revealed localization of the PLA signals representing the VEGFR2-ephrinB2 complex (magenta) to postsynaptic sites (indicated by arrowheads). Scale bar: 10 µm. (D–E) Primary wild type hippocampal neurons at 14 DIV were stimulated with VEGF for 30 min and PLA was performed using the EphB4-Fc and an anti-VEGFR2 specific antibody. VEGFR2-ephrinB2 complex (PLA signal, magenta) localizes to early endosomes labeled using antibodies against the early endosomal compartment protein EEA1 (green) and stimulation with VEGF leads to increased localization of the VEGFR2-ephrinB2 complex to early endosomes. Representative images are shown in (D) and quantification of the percentage of PLA puncta colocalizing with EEA1 labeled endosomes is shown in (E) (n = 3 experiments). Scale bar: 10 µm. Data are represented as mean ± SEM. *p<0.05.