P creb

Human Aβ_1–42 was purchased from EMD Millipore Corporation (Billerica, MA, USA). Ori was obtained from Chengdu Must Bio-Technology Company (Sichuan, China). Neurobasal media and B27 were bought from Life Technologies Corporation. MAP-2 antibody was purchased from Abcam (Cambridge, MA, USA). COX-2 antibody was bought from Santa Cruz Biotechnology (Santa Cruz, USA). PSD-95 antibody, synaptophysin antibody and CREB antibody were obtained from CST (Cell Signaling Technology, USA). 4', 6'-diamidino-2-phenylindole (DAPI), VDAC1, BDNF, p-TrkB, TrkB, p-CREB and β-actin antibodies were purchased from Bioworld Technology (Bioworld, USA). Lamin B1 antibody and HRP-conjugated secondary antibodies were also obtained from Bioworld.

3,6’-Disinapoyl sucrose and TSFA were isolated from Radix Polygalae, identified by a combination of spectral methods (UV, IR, MS, and NMR) and purified by HPLC (purity >90%). U0126 and LY294002 were purchased from Selleck Chemicals LLC (Houston, TX, USA). Dulbecco’s modified Eagle’s medium (nutrient mixture F-12; DMEM/F-12) was obtained from Gibco (Carlsbad, CA, USA). Fetal bovine serum (FBS) was supplied by Hyclone (Logan, UT, USA). Polyclonal rabbit antibodies against BDNF, CREB, pCREB, CRTC1, and β-actin were all provided by Bioworld Technology (Atlanta, GA, USA) or Abcam (Burlingame, CA, USA).

Melan-a cell lysates were mixed with sample buffer (250 mM Tris-HCl (pH 6.8), 0.5 M DTT, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% 2-mercaptoethanol), and denatured at 100 °C for 5 min using a standard protocol. A 10% of SDS-PAGE was used to separate the sample proteins (50 µg). Following electrotransfer to nitrocellulose membranes (Whatman, Dassel, Germany), the membranes were immersed overnight in a mixture containing antibodies and 5% skim milk. Primary antibodies, such as anti-tyrosinase, anti-TRP-1, anti-TRP2, anti-MITF, p-CREB, and total CREB and β-actin, were from Bioworld Technology (St. Louis Park, MN, USA). Secondary antibodies (anti-goat IgG-horse radish peroxidase (HRP) and anti-mouse IgG-HRP) were purchased from Santa Cruz. The resulting reaction was exposed using an ECL solution system (Perkin Elmer).