Figure 1

Inhibition of TrkB suppressed NRG1 induced activation of NR2B in primary cortical neurons. (Ai) Time dependent effect of NRG1 on ErbB₄ and TrkB receptor activation. NRG1 (5 nM) was applied at DIV4 for indicated time course. (Aii) pErbB₄ values were normalized against total ErbB₄ levels and (Aiii) pTrkB values were normalized against total TrkB levels. Data represent mean ± SEM. (n = 3). *P < 0.01 vs control (CON). (Bi) Trk inhibitor K252a blocks the stimulatory effect of NRG1 on NR2B activation, but not ErbB₄ activation in cortical neurons. Cortical neurons were pretreated with 100 nM K252a for 30 min, followed by NRG1 for 5 min. (Bii) Quantification of normalized pNR2B and (Biii) pErbB₄. Data represent mean ± SEM. (n = 5). *P < 0.01 vs control; ^$P< 0.01 vs NRG1 treatment. (Biv) The specificity of the pErbB4 antibody was tested in cortical neurons. Immunoprecipitation (IP) with the anti-ErbB4 antibody or control IgG antibody followed by blotting with the anti-pErbB4, anti-pTrkB or anti-ErbB4 antibody was performed in neurons (DIV4) after NRG1 stimulation. (C) Endogenous TrkB was decreased after TrkB-siRNA transfection compared to scrambled siRNA. siRNA was transfected 48 h before lysates were collected. Data represent mean ± SEM. (n = 3). *P < 0.05. (D) NRG1-induced NR2B activation was reduced in TrkB-siRNA transfected cultures. Data represent mean ± SEM. (n = 3). *P < 0.05 vs scrambled siRNA group; ^$P < 0.05 vs NRG1 treated scrambled siRNA group. (E) Immunoblotting analysis of NRG1-induced NR2B activation in primary cortical neurons isolated from WT and TrkB−/− mice. Data represent mean ± SEM. (n = 3). *P < 0.05 vs WT control; ^$P < 0.05 vs WT NRG1 treatment.