Radioimmunoprecipitation assay buffer

Following experimental exposure, rat pups (n=5 per group) were sacrificed and western blot analysis was performed as previously described (20 (link)). In brief, hippocampus tissues were homogenized in cold radioimmunoprecipitation assay buffer (Applygen Technologies Inc., Beijing, China), and the quantity of protein in the supernatants was determined using a bicinchoninic acid protein assay kit (Applygen Technologies Inc.). Protein samples (60 mg protein/lane) were separated by 8 or 10% SDS-PAGE. Following transfer to nitrocellulose membranes, the blots were probed using the following primary antibodies: Rabbit anti-Akt (Ser⁴⁷³; cat. no. 9272), anti-phosphorylated (p)-Akt (Ser⁴⁷³; cat. no. 9271), anti-GSK-3β (Ser⁹; cat. no. 9315), and anti-p-GSK-3β (Ser⁹; cat. no. 9336) antibodies (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA); and rabbit anti-B-cell CLL/lymphoma 2 (Bcl-2; cat. no. sc-783), anti-Bcl-2-associated X protein (Bax; cat. no. sc-526) and anti-caspase-3 (cat. no. 7148) antibodies (1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Fluorescent secondary antibodies (1:10,000; cat. nos. 926–32211 and 926–32210; LI-COR, Inc., Lincoln, NE, USA) were used to detect the binding of primary antibodies. Proteins were visualized by scanning the membrane on an Odyssey Infrared Imaging System (version 3.0; LI-COR, Inc.).

Western blot analysis was performed as previously described (11 (link)). In brief, the diaphragms were rinsed with ice-cold PBS, harvested with radioimmunoprecipitation assay buffer (Applygen, Beijing, China) and quantified using a bicinchoninic acid protein assay (WellBiology, Changsha, China). Total protein (30 µg/lane) was separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h at room temperature and probed with antibody against myostatin (cat no. 19142-1-AP) and β-actin (cat no. 20536-1-AP; 1:1,000; Proteintech, Chicago, IL, USA) at 4°C overnight, followed by incubation with a secondary antibody (cat no. SA00001-2; 1:5,000; Proteintech, Chicago, IL, USA) conjugated to horseradish peroxidase at room temperature for 1 h. Immunoreactivity was detected by enhanced chemiluminescent agent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The protein expression levels were quantitatively analyzed and normalized against the β-actin loading control.

At the end of experiment, all rats were sacrificed with 10% chloral hydrate solution (3.5 ml/kg; i.p.). Hippocampus and prefrontal cortex tissues were collected and lysed with radioimmunoprecipitation assay buffer (Applygen Technologies, Inc., Beijing, China), schizolysised for 20 min on ice and centrifuged at 12,000 × g for 10 min at 4°C. Protein samples were heated at 95°C for 8 min, separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membrane were blocked for 2 h in TBST (25 mM Tris, 140 mM NaCl, 27 mM KCl and 0.02% Tween 20) containing 5% bovine serum albumin and incubated with primary antibodies specific for BDNF (ab108319, 1:200)(Abcam, Cambridge, MA, USA), TrkB (BS1431, 1:500), ERK (AP0491, 1:500), pERK (BS4621, 1:500), Akt (BS1502, 1:500), PI3K (BS3678, 1:500), pGSK3β (BS4084, 1:500) and GSK3β (BS1402, 1:500; Bioworld Technology, Inc., St. Louis Park, MN, USA), pCREB (#9198, 1:1,000) and CREB (#9197, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C over night. Following three washes with TBST, membranes were incubated for 1 h at room temperature with horseradish peroxidase-labeled secondary antibodies (BS13278, 1:5,000; Bioworld Technology, Inc.), washed with TBST three times. Blots were developed using a electrochemiluminescence system (UVP LLC, Upland, CA, USA).