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. 2014 Nov 5;34(45):14995–15008. doi: 10.1523/JNEUROSCI.2228-14.2014

Figure 4.

Figure 4.

Akt-mediated Girdin S1416 phosphorylation regulates BDNF-induced NR2B phosphorylation of NMDA receptor through Src kinase. A, B, Time-course changes in the levels of phosphorylated NR2A (A) and NR2B (B) after BDNF (50 ng/ml) application in cultured E15–E16 mouse hippocampal neurons (DIV 14). Data are expressed as mean ± SEM (n = 4 in each time point for A; n = 5 in each time point for B). One-way ANOVA, F(3,16) = 6.021, p < 0.01 for B. *p < 0.05 compared with control (Tukey–Kramer post hoc test). C–E, Western blot analyses showing the levels of phosphorylated Akt, Girdin, and NR2B after BDNF application. Cultured E15–E16 mouse hippocampal neurons (DIV 14) were treated with PBS (control) or BDNF (50 ng/ml) for 15 min. C, No changes in BDNF-induced Akt phosphorylation in Girdin+/+ and GirdinSA/SA neurons. D, E, BDNF-induced phosphorylation of Girdin S1416 and NR2B was abolished in GirdinSA/SA neurons. Data are expressed as mean ± SEM (n = 5 for Girdin+/+; n = 5 for GirdinSA/SA). Two-way ANOVA; C, genotype, F(1,16) = 0.799, p = 0.3846; BDNF, F(1,16) = 16.981, p < 0.01; genotypes × BDNF, F(1,16) = 0.041, p = 0.8420; D, genotype, F(1,16) = 812.318, p < 0.01; BDNF, F(1,16) = 27.516, p < 0.01; genotypes × BDNF, F(1,16) = 27.516, p < 0.01; E, genotype, F(1,16) = 29.733, p < 0.01; BDNF, F(1,16) = 5.069, p < 0.05; genotypes × BDNF, F(1,16) = 14.556, p < 0.01. **p < 0.01 compared with corresponding control. N.D., not detectable. F, Interaction of Girdin with NR2B and Src, but not Fyn. Coimmunoprecipitation among Girdin, NR2B, and Src was performed in HEK293T cells that overexpressed Girdin-V5 and NR2B together with either Src-GFP or Fyn-GFP.