Sc 20156

Proteins from the liver homogenate and hepatic fractions were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, transferred to nitrocellulose membranes, and blocked for 1 h in Tris-Buffered Saline and Tween 20 (TBST) buffer (20 mmol/L Tris, pH 7.5, 500 mmol/L NaCl, 0.5% Tween 20) with 5% non-fat milk. Anti-TPH1 (ab52954, Abcam, Cambridge, U.K.) (1/3500 in TBST) or anti-MAOa (sc-20156, Santa Cruz Biotechnology, Inc, Santa Cruz, CA) (1/1000 in TBST) was added as primary antibody and incubated overnight at 4°C. As loading controls for liver homogenates, membranes were incubated with mouse anti-tubulin antibody (ab 56676 Abcam, UK) diluted 1/1000, for mitochondrial fractions was used as control protein of mitochondrial origin, rabbit anti-VDAC1/Porin antibody (ab15895; Abcam, UK) diluted 1/1000. After washing, membranes were incubated with the appropriate secondary antibodies conjugated to alkaline phosphatase (sc2315, Santa Cruz Biotechnology, Inc.)1/5000. Bands were revealed using the AP conjugate substrate kit (Bio-Rad, CA). Densitometric analysis was performed using the Image Lab Software (v 3.0, Bio-Rad, CA).

The brain tissues were sonicated on ice in 1x RIPA buffer with proteinase inhibitors (Protease Inhibitor Cocktail Set III, Animal-Free-Calbiochem, Millipore, Billerica, MA, USA) three times for 5 s each. Small aliquots of the extracts were retained for protein determination by using bicinchoninic acid assay (Thermo scientific, Waltham, MA, USA), with bovine serum albumin as the standard. Thereafter, equal amounts of protein (20–40 μg) were separated through SDS-polyacrylamide gel electrophoresis (10%–12.5% polyacrylamide) and transferred onto a polyvinylidene fluoride (Millipore) membrane; the signals were measured using an enhanced chemiluminescence detection kit (Millipore). The antibodies against TH (ab6211, Abcam, Cambridge, UK), TH-phosphoSer40 (AB5935, Millipore), MAO-A (SC-20156, Santa Cruz, Dallas, TX, USA), and NAO-B (SC-18401, Santa Cruz) were used to detect the protein expression levels and activities. The signals were detected on a ChemiDoc™ XRS+ Image System (Bio-Rad Laboratories, Hercules, CA, USA). The blots were then stripped and reprobed using anti-GAPDH (NB300-221, Novus Biologicals, Littleton, CO, USA), anti-beta-actin (NB-600-501, Novus Biological), or anti-TH (ab137869, Abcam) antibodies for quantitative control analysis.