Thermo scientific supersignal west pico chemiluminescent substrate solution

Cells transiently expressing wild-type or mutant human DAT were washed with cold PBS and incubated with sulpho-NHS-SS-biotin (1 mg/ml PBS; Pierce Biotechnology) for 60 min at 4°C, before incubation with 100 mM glycine in PBS for 20 min and extensive washing. The washed cells were lysed in mammalian protein extraction reagents (Thermo Scientific) supplemented with a protease inhibitor cocktail (Thermo Scientific) for 10 min at 21°C and transferred into eppendorf vials. The vials were incubated for 60 min on ice with vortexing every 5 min. The lysate was then centrifuged at 14 000 g for 15 min at 4°C and supernatant was collected for preparing total lysates and separating biotinylated cell surface proteins. The biotinylated proteins were separated with immobilized monomeric NeutrAvidin (Thermo Scientific) and eluted with SDS-PAGE sample buffer. The total lysates and biotinylated proteins were resolved on 8% Tris-glycine mini gels and probed with polyclonal anti-DAT antibody against the C-terminal of DAT (Millipore), before horseradish peroxidase-conjugated goat anti-rabbit antibody. Polyclonal anti-β-actin antibody (Sigma-Aldrich) was used as an internal control for loading. The transporter signal was visualized using Thermo Scientific SuperSignal® West Pico Chemiluminescent Substrate solution (Thermo Scientific).