Mao b assay kit

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 100 units/mL penicillin and 100 mg/mL streptomycin are purchased from Gibco (Thermo Fisher ScientificWaltham, MA, USA). 1-Methyl-4-pheynl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride, 1-Methyl-4-phenylpyridinium (MPP⁺) iodide, 3-methyladenine (3-MA) and ropinirole were purchased from Sigma Chemical Co (St. Louis, MO, USA). Wortmannin (Wort) and bafilomycin A1 (Baf) are from Abcam (MA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-AMPK, anti-phosphorylated AMPK (p-AMPK), anti-ULK, anti-phosphorylated ULK555 (p-ULK555), anti-Erk, anti-phosphorylated Erk (p-Erk), anti-mTOR, anti- phosphorylated mTOR (p-mTOR), anti-LC3B, anti-tyrosine hydroxylase (TH), anti-α-synuclein primary antibodies sourced from rabbit, anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody, the Erk inhibitor U0126, the AMPK siRNA, AMPK siRNA control were purchased from Cell Signaling Technology (Boston, MA, USA). An autophagy Tandem Sensor RFP-GFP-LC3B kit was from Thermo Fisher Scientific. MTT assay kit Z-Cytox was from DAEILLAB Co, Ltd (Seoul, Korea). Dopamine ELISA kit was from Abnova (Taipei City, Taiwan) and the MAO-B assay kit was supplied by Promega (Woods Hollow Road Madison, WI, USA). All other chemicals were of the highest grade and were found from commercial resources.

The MAO-B activity of the ST and SN was determined by a MAO-B assay kit from Promega according to the manufacturer’s instructions. The kit applied a homogeneous luminescent method for detecting MAO activity. The assay includes 2 steps. First, the MAO-B substrate was added to MAO-B enzyme-containing material (ST and SN samples) to generate methyl ester luciferin; second, the produced methyl ester luciferin reacted with esterase and luciferase to produce light. The MAO-B activity was directly proportional to the amount of developed light. The luminescent signal was measured using an Infinite M1000 multimode microplate reader (TECAN, Männedorf, Switzerland). MAO-B activity was expressed as relative light units (RLU)/mg protein.