Journal: The Journal of Neuroscience
Article Title: Neuronal Depolarization Controls Brain-Derived Neurotrophic Factor-Induced Upregulation of NR2C NMDA Receptor via Calcineurin Signaling
Figure Lengend Snippet: 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
Article Snippet: Slices were treated with or without 1 μ m FK506 for 96 h, and 0.5 vol of the culture medium was changed every 2 d. Reagents and inhibitors were purchased from the following sources: BDNF (Peprotech, London, UK); neurotrophin-3 (NT-3), NT-4, and nerve growth factor (NGF) (Alomone Laboratories, Jerusalem, Israel); neuregulin-β (NeoMarkers, Fremont, CA); nifedipine (Nacalai Tesque, Kyoto, Japan); KN62 (Tocris, Bristol, UK); and U0126, SB203580, FK506, K252a, , PD98059, cyclosporin A, rapamycin, and bisindolylmaleimide I (Calbiochem, San Diego, CA).
Techniques: Activation Assay, Cell Culture, Isolation, Immunoprecipitation, Knock-Out, Mouse Assay, Staining