Figure 2.
BDNF-induced NR2C upregulation by TrkB activation. Cells were cultured in low KCl and treated with or without BDNF (100 ng/ml in A and 50 ng/ml in B-D and F) for 96 h. A, Levels of indicated mRNAs were quantitated by RNA blotting (n = 4). B, Cell lysates (40 μg) were immunoblotted with anti-panNR2 antibody or anti-NR1 antibody. Molecular sizes (kilodaltons) of protein makers are indicated on the left. C, P2 membrane fractions were isolated, solubilized, and immunoprecipitated (IP) with anti-NR1 antibody, followed by immnoblotting with anti-panNR2 antibody. D, Cell-surface proteins were biotinylated with Sulfo-NHS-SS-Biotin. Cell lysates were solubilized, precipitated with NeutrAvidin beads, and immunoblotted with anti-panNR2 antibody. E, Cells were cultured in low KCl and treated with BDNF, NGF, NT-3, or NT-4 (50 ng/ml each) for 96 h. Levels of NR2C mRNA were quantitated (n = 4). F, Granule cells were prepared from TrkB-/- knock-out mice(KO) and their wild-type (WT) littermates. NR2C and TrkB mRNAs were analyzed by RNA blotting. Ethidium bromide-stained 18s rRNA is also indicated. *p < 0.05; **p < 0.01. Error bars indicate SEM.