Anti p trkb

Following primary antibodies were used in the present study: anti-GAPDH (1:5000; arigo, ARG10112), anti-TH (1:1000; BD biosciences, 612300), anti-α-synuclein (1:1000; BD bioscicences, 610786), anti-p-TrkB (1:1000; abcam, ab229908), anti-trkB (1:1000; Cell Signaling Technology, 4603 T), anti-LC3A/B (1:1000; Cell Signaling Technology, 12741S), anti-P62 (1:1000; proteintech, 18420-1-AP), anti-beclin-1 (1:1000; Cell Signaling Technology, 3738S), anti-p-ERK1/2 (1:1000; Cell Signaling Technology, 4370S), anti-ERK1/2 (1:1000; Cell Signaling Technology, 4695), anti-p-LKB1 (1:1000; Cell Signaling Technology, 3482S), anti-LKB1 (1:1000; Cell Signaling Technology, 3047), anti-p-AMPK (1:1000; Cell Signaling Technology, 2535S), anti-p-mTOR (1:1000; Cell Signaling Technology, 5536T), anti-AMPK (1:1000; Cell Signaling Technology, 2532) and anti-p-ULK1 (1:1000; Cell Signaling Technology, 5869).

The brain tissue samples and SH-SY5Y cells were lysed and homogenized in cold RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, 50 mM Tris-Cl, pH 7.5). The solution was centrifuged at 14,000 rpm for 15 min at 4 °C and the supernatant protein concentration was determined using a commercial BCA assay kit (Thermo Scientific, Waltham, MA, USA). To separate proteins, electrophoresis was conducted, and they were transferred to PVDF membranes. The anti-BDNF, TrkB, p-CREB, CREB, p-ERK, and ERK antibodies (1:1000; Cell Signaling), and anti-p-TrkB (1:1000; Abcam), and anti-GAPDH (1:5000; Cell Signaling) were applied during an overnight incubation at 4 °C. Following this, membranes were incubated with appropriate secondary antibodies at room temperature (RT). The band intensities were visualized using the ECL Western Blotting Detection System (Amersham Biosciences, Pittsburgh, PA, USA) and analyzed using a luminescent image analyzer LAS-4000 (GE Healthcare, Uppsala, Sweden). Densitometric analysis of Western blotting data was performed using the Image J software (version 1.53) (NIH, Bethesda, MD, USA).