Mab369

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.

SDS-PAGE was performed on a 5–20% gradient gel (E-R520L, ATTO) and separated proteins were transferred onto a polyvinyldifluoridine membrane (Immobilon, Millipore). After blocking with 4% non-fat dry milk and 0.1% Triton X-100 in TBS, membranes were incubated with rat anti-DAT (cat no. MAB369, Millipore; 1:4000 dilution) or rabbit anti-PTPRZ-S prepared in our laboratory [19 (link)] (1:3000 dilution), followed by a treatment with HRP-conjugated anti-rabbit IgG (cat no. NA9340V, GE Healthcare; 1:3000 dilution) or anti-rat IgG (cat no. 1132-036-072, Jackson Immunoresearch; 1:4000 dilution), respectively. After washing, the blots were incubated with a chemiluminescent substrate (Luminata forte western HRP substrate, Millipore), and the chemiluminescence signal was immediately detected using a CCD camera system (Ez-capture MG, ATTO Bioscience & Technology) for a length of time sufficient to ensure detection without saturation.

For the immunofluorescence evaluation of DAT in CPu, free-floating sections were rinsed in PB 0.1M, blocked, and permeabilized in 3% normal goat serum and 0.3% Triton X-100 in 0.1 M PB at room temperature (3 h), followed by incubation in the same solution with the primary antibody (monoclonal rat anti-DAT; 1:1,000, MAB369, Millipore, CA, United States) at 4°C (2-nights). Then, sections were rinsed three times in PB 0.1 M and incubated with the biotinylated secondary antibody (biotinylated goat anti-rat; 1:200) at room temperature (2 h), followed by incubation with AlexaFlour^® 488-labeled streptavidin (1:500, Jackson ImmunoResearch Europe, Newmarket, United Kingdom) at room temperature (1 h). Afterward, sections were rinsed in PB 0.1 M and mounted onto super-frost glass slides using Mowiol^® mounting medium (Costa et al., 2017 (link)). Omission of either the primary or secondary antibodies served as negative control and yielded no labeling (data not shown).