Anti bdnf sc 546

Protein expression was assessed by Western blotting. Cells were harvested from the culture plates and total cellular proteins were extracted by lysis buffer containing 1% Triton X-100, 1% proteasome inhibitor cocktail (Sigma-Aldrich). Lysates were cleared by centrifugation at 13,000 g for 20 min and protein concentrations were measured using the BC Assay Protein Quantitation Kit (Uptima, Oakland, CA). Total protein lysates (50 μg) were fractionated on 15% SDS-PAGE then transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Vélizy-Villacoublay, France). Membranes were blocked for 1 h in PBST with 2.5% BSA, and then incubated with primary antibodies overnight at 4 °C, rabbit polyclonal anti-BDNF (sc-546, Santa Cruz [51 (link)]) at 1:200 and mouse monoclonal anti-α-tubulin (Clone DM1A, Sigma-Aldrich) at 1:10,000. Incubation with secondary antibodies conjugated to infrared fluorophores (goat anti-rabbit IgG Dylight 800 and anti-mouse IgG Dylight 680 at 1:10,000 from Thermo Scientific) was performed for 1 h. An Odyssey infrared imaging system (LI-COR, Bad Homburg, Germany) was used to scan membranes at a wavelength of 680 nm (anti-mouse) or 800 nm (anti-rabbit). Data were analyzed with Image Studio 1.1 software (Li-COR).

Escitalopram oxalate (Pangbourne, UK), and paroxetine HCl (Holzkirchen, DE) were gifts from Sandoz. The antibodies for the Western blot analyses were obtained from the following sources: anti-phospho-mTOR (Ser2448, #2971), anti-mTOR (#2972), anti-phospho-Akt (Ser473, #9271), anti-Akt (#9272), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, #9101), anti-p44/42 MAPK (ERK1/2, #4695), anti-phospho-4E-BP-1 (Thr37/46, #2855), anti-4E-BP-1 (#9452), anti-phospho-eIF4B (Ser422, #3591), anti-eIF4B (#3592), anti-phospho-S6 (Ser240/244, #2251), anti-S6 (#2217), anti-phospho-p70S6 K (Thr389, #9205), and anti-p70S6 K (#9202) from Cell Signaling Technology (Beverly, MA, USA); anti-BDNF (sc-546) and goat anti-rabbit IgG-horseradish-peroxide conjugates (sc-2004) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and monoclonal anti-α-tubulin (T9026) and anti-mouse IgG peroxidase conjugates (A4416) from Sigma (St. Louis, MO, USA).

Western blots were performed as described previously [43 ]. Briefly, the dissected mPFC samples were homogenized in a lysis buffer (Beyotime Biotechnology, Shanghai, China) with 1% phenylmethylsulfonyl fluoride (Sangon Biotech, Shanghai, China) and 1 × PhosSTOP phosphatase inhibitor cocktail (Roche Applied Science, Penzberg, Germany). The extracted proteins were separated on a 15% SDS-PAGE gel and transferred to PVDF membrane (Millipore, Massachusetts, USA). The membrane was blocked with 5% non-fat milk powder in TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween20), followed by incubation with the following primary antibodies diluted in a blocking solution overnight at 4 °C: anti-BDNF (sc-546, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), anti-FoxO1 (1:500, #2880, Cell Signaling Technology, Danvers, MA, USA), or anti-β-actin antibody (1:1000, #4970, Cell Signaling Technology, Danvers, MA, USA). After washing, the membrane was incubated with IRDye 680LT donkey anti-rabbit IgG secondary antibodies (1:5000; 926–68,023, Li-COR Biosciences, Lincoln, NE, USA). The fluorescence was visualized and analyzed using an Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA).