Anti trkb

The protein samples (50 µg of protein) were prepared from micro-dissected hippocampus and separated by 12% SDS-PAGE gel. Proteins were transferred to nitrocellulose membranes using a Bio-Rad Miniprotein III System wet transfer unit for 1 h at 4 °C. Transfer membranes were then incubated with blocking solution (5% nonfat dried milk dissolved in TBST buffer, pH 7.5) for 1 h at room temperature, then incubated with anti-BDNF (1:1000, Abcam), anti-PKA(1:1000, Imagenex), anti-CREB (1:1000, Cell Signaling), anti-pCREB (Ser133, 1:1000, Cell Signaling), anti-MeCP2 (1:2000, Cell Signaling), anti-pMeCP2 (Ser421, 1:1000, Abgent), anti-TrkB (1:1000, Millipore) and anti-pTrkB (Tyr706, 1:1000, Cell Signaling) overnight at 4 °C. Membranes were incubated with secondary antibodies for 1 hat 4 °C. Signal detection was performed with an enhanced chemiluminescence kit (Amersham Biosciences) and quantitated by using the GS-710 Calibrated Image Densitometer (Bio-Rad).

Hippocampal neurons were plated at a density of 10⁶ cells/cm². At 7DIV neurons were incubated in B27-free neurobasal medium and then stimulated with BDNF 50 ng/mL (Alomone) for 1 h. Next, neurons were washed and lysed in immunoprecipitation assay buffer (20 mM Tris, 150 mM NaCl, 10% glycerol, 1% Igepal, 2 mM EDTA) supplemented with inhibitors (1 mM PMSF, 1 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mM Na₃VO₄, and 50 mM NaF). Cell lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. Protein quantification was performed using the Pierce^® BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). For immunoprecipitation assays, 300–500 µg of total lysates were incubated with 2 µg of anti-c-Abl (K12 Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-TrkB (Millipore) antibodies overnight at 4 °C. Complexes were isolated using protein G-Plus agarose (Santa Cruz cat# sc-2002). Immunocomplexes were subjected to SDS-PAGE and transferred to nitrocellulose membranes (Fisher Thermo Scientific) and analyzed by western blot using anti-TrkB (1:1000), anti-c-Abl (1:1000), and anti-GAPDH (1:5000) antibodies.

Commercial antibodies included: anti-TrkB (BD Biosciences, New Jersey, NJ, USA; #cat 610102), anti-TrkB (Millipore, Burlington, MA, USA; #cat 07-225), anti-phospho-TrkB (Tyr 515) (Sigma, St Louis, MO, USA; #cat SAB4503785), c-Abl (Sigma #cat A5844), anti-c-Abl (Santa Cruz Biotechnology, Dallas, TX, USA; #cat K-12), anti-c-Abl (Santa Cruz Biotechnology, #cat 24-11), anti-phospho-c-Abl (Tyr 412) (Sigma, #cat C5240), anti-PLC-γ (Cell Signaling, Danvers, MA, USA; #cat 2822), anti-phospho-PLC-γ (Tyr 783) (Cell Signaling, #cat 2821), anti-AKT (Cell Signaling, #cat 9272), anti-phospho-AKT (Ser 473) (Cell Signaling, #cat 9291), anti-ERK (Cell Signaling, #cat 9102), anti-phospho-MAPK (Erk1/2; p42/44; Thr202/Tyr204) (Cell Signaling, #cat 9101), anti-Flag (Sigma, #cat F3040), anti-GAPDH (Santa Cruz, #cat 6C5), β-III Tubulin (Sigma, #cat T8578), anti-MAP2 (Sigma, #cat M3696), anti-phospho-CrkII (Tyr221) (Cell Signaling, #cat 3491), anti-CrkII (Cell Signaling, #cat 3492), Secondary Antibody: Goat anti-Mouse IgG (H + L) and Goat anti-Rabbit IgG (H + L), HRP (Invitrogen, Carlsbad, CA, USA, #cat 31430 and #cat 31460) and secondary antibody Alexa-555, Alexa-488, Alexa-633 and Alexa 547 (1:1000) (Invitrogen, Carlsbad, CA, USA).