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. 2017 Oct 3;21(1):84–96. doi: 10.1016/j.celrep.2017.09.019

Figure 6.

Figure 6

Ser-9 of EphrinB2 Is Essential for the ApoER2-Mediated Insertion of AMPA Receptors at the Post-synaptic Membrane

(A–C) The macromolecular complex consisting of ApoER2, GRIP1, GluR2, and ephrinB2 is disrupted in ephrinB2 S-9 > A mice. Ser-9 of ephrinB2 is necessary for the binding of GRIP1 and GluR2 to ephrinB2. Wild-type (+/+) and ephrinB2 S-9 > A (efnB2 S-9 > A) total brain lysates were immunoprecipitated by using an ephrinB2 antibody or an unrelated goat antibody (Ctrl), and binding of GRIP1 and GluR2 was investigated by western blot (A). The interaction between ApoER2 and GRIP1 is lost in ephrinB2 S-9 > A mutant mice. Wild-type (+/+) and ephrinB2 S-9 > A knockin (efnB2 S-9 > A) brain lysates were immunoprecipitated using anti-ApoER2 antibody or an unrelated rabbit antibody (Ctrl), and the interaction with GRIP1 was analyzed by western blot (B). ApoER2 and GluR2 co-immunoprecipitation in mouse brain lysates is dependent on Ser-9 of ephrinB2. Total brain lysates from adult wild-type (+/+) and ephrinB2 S-9 > A knockin (efnB2 S-9 > A) mice were immunoprecipitated with anti-GluR2 antibody or an unrelated mouse antibody (Ctrl) and analyzed by western blot for ApoER2 and GluR2 (C).

(D–G) Ser-9 of ephrinB2 is required for AMPA receptor membrane insertion and functionally interacts with ApoER2 upon stimulation with KCl and Reelin. Wild-type (+/+) and ephrinB2 S-9 > A knockin (efnB2 S-9 > A) neurons were subjected to AMPA receptor membrane insertion assays upon stimulation with 10 mM KCl or concentrated Reelin (Reln) supernatant. Fluorescent images of newly inserted GluR2 staining (D). Fluorescent images of newly inserted GluR2 in wild-type (+/+) and ApoER2+/−; ephrinB2S-9>A/+ compound neurons upon KCl or Reelin (Reln) stimulation (E). Shown are quantification of newly inserted GluR2 intensities in wild-type (+/+) and ephrinB2 S-9 > A (efnB2 S-9 > A) neurons (n = 10) (F) and quantification of fluorescence intensities in wild-type, ApoER2+/−, ephrinB2S-9>A/+, and compound neurons (n = 3–4) (G).

Scale bars in (D) and (E) represent 5 μm. Bar graphs show mean ± SEM (shown as error bars). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S5 and S6.