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. 2024 May 17;10(20):eadi7024. doi: 10.1126/sciadv.adi7024

Fig. 3. Presynaptic EphB2 interacts with Doc2B and phosphorylates Doc2B at Y36.

Fig. 3.

(A) Immunostaining against Doc2B (green) and EphB2 (red) in the mouse hippocampus region. Nuclear DNA was labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 500 μm. (B) Separation of pre- and postsynaptic densities from purified synaptosomes. Syn, synaptosome; Pre, presynaptic elements; Post, postsynaptic elements; Extra, extrajunctional synaptic elements. (C and D) Western blot analysis of Doc2B-EphB2 interaction following cotransfection of mCherry-tagged Doc2B and Flag-tagged EphB2 in HEK293 cells. Cell lysates were immunoprecipitated by anti-mCherry antibody (C) or anti-Flag antibody (D), followed by immunoblotting with indicated antibody. (E) Schematic representation of EphB2 constructs. (F and G) Western blot analysis of Doc2B-EphB2 interaction following cotransfection of various combinations of plasmids containing EphB2 FL, EphB2-∆SP, EphB2-∆KSP, and/or mCherry-tagged Doc2B (F), EphB2 WT, KR, and/or mCherry-tagged Doc2B (G) in HEK293 cells. (H and I) In vitro phosphorylation assay using sumo-tagged EphB2 and GST-tagged Doc2B fragments (H) and GST-tagged Doc2B Mid or Y36F (I) in the presence or absence of ATP and APase. Asterisks show bands of GST-Doc2B Mid, Mid-L, and FL, respectively. CBB, Coomassie Brilliant Blue staining. (J and K) Western blot analysis of Doc2B phosphorylation following cotransfection of mCherry-tagged Doc2B WT or Y36F and/or EphB2 (J) and EphB2 WT, KR, or YYEE and/or mCherry-tagged Doc2B (K) in HEK293 cells. (L) Detection of Doc2B phosphorylation in cultured mouse cortex neurons by application of Fc (control) or preclustered Ephrin-B3-Fc. (M and N) Western blot analysis of Doc2B phosphorylation in EphB2+/+ or EphB2−/− mouse brains (M) in Doc2A-deficient neurons infected with Doc2B KD or control virus (N). Tubulin used as the reference protein. Data are presented as means ± SEM (n = 3). Statistical significance and P values were determined by Student’s t test (**P < 0.01; ****P < 0.0001).