Ab5935

Rat anti-DAT (MAB369; Millipore; 1:2000 dilution) or rabbit monoclonal anti-DAT (AB184451, Abcam, 1:1000 dilution for Fig. S2, A and B only), rabbit anti-TH (AB152, Millipore, 1:10,000 dilution), rabbit anti-pSer40 TH (AB5935, Millipore, 1:5000 dilution), mouse antitransferrin receptor (clone H68.4, Thermo Fisher; 13-6800). Secondary antibodies conjugated to horseradish peroxidase were all from Jackson ImmunoResearch, and immunoreactive bands were visualized by chemiluminescence using SuperSignal West Dura (Thermo Scientific). Nonsaturating immunoreactive bands were detected using either a VersaDoc 5000MP or a ChemiDoc imaging station (Bio-Rad) and were quantified using Quantity One software (Bio-Rad). Surface DAT was calculated by normalizing the biotinylated DAT signal to its corresponding amount of total DAT in that sample, detected in parallel from the same exposure of the same immunoblot. Raw surface DAT values are expressed as %total. For most experiments, DAT surface values following drug treatment were normalized to vehicle-treated samples, obtained from the contralateral hemislice in parallel. Surface DAT bands, and their corresponding total lysate, shown for each experiment were taken from the same exposure of the same immunoblot, and brightness/contrast levels were set identically for all blots. Boxed bands were cropped and rearranged for presentation purposes only.

Brain-tissue samples were collected using 2-deoxy-d-glucose, ethylene glycol tetra-acetic acid (EGTA), and reduced glutathione. Western blot and immunofluorescence analyses involved primary antibodies; primary monoclonal rat anti-tyrosine hydroxylase (T1299, Sigma-Aldrich, St. Louis, MI, USA), monoclonal rabbit anti-NPY5R (AB133757, Abcam, Cambridge, UK), polyclonal rabbit AMPKα1/2 (H-300) (sc-25792, Santa Cruz, CA, USA), polyclonal rabbit anti-phospho AMPKα (THR172, Millipore, Burlington, MA, USA), monoclonal mouse anti-β-actin (A5441, Sigma-Aldrich, St. Louis, MI, USA), and polyclonal rabbit anti-tyrosine hydroxylase phosphoSer40 (AB5935, Millipore, Burlington, MA, USA). Primary antibodies in Western blot analysis were detected using polyclonal goat anti-mouse IgG HRP (HAF007, R&D Systems, Minneapolis, MN, USA) and polyclonal goat anti-rabbit IgG (H + L) HRP (65-6120, Invitrogen, Waltham, MA, USA), whereas Alexa Fluor 488 conjugated with goat anti-rabbit IgG (H + L) secondary antibody (A11008, Thermo Fisher Scientific, Waltham, MA, USA) and CY3 conjugated with goat anti-mouse IgG (H + L) secondary antibody (A10521, Thermo Fisher Scientific, Waltham, MA, USA) were used in immunofluorescence.

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.