[3H]CFT binding assay

Prior to [³H]CFT binding assay, LLC-PK₁ cells stably transfected with hDAT-WT were treated with various concentrations (1 µM, 3 µM, 10 µM, 30 µM, 50 µM and 100 µM) of crosslinking agent CuP diluted in PBS for 5 minutes at 21°C, as indicated in text. N-ethylamine was not used here to quench residual CuP because it greatly affected the following [³H]CFT binding assay (data now shown). Crosslinker CuP was then thoroughly washed off and cells were dislodged from cell-culture plates by trypsinization. Saturation analysis of [³H]CFT ((2β-carbomethoxy-3β-[4-fluorophenyl]-tropane, 85.9 Ci/mmole, Perkin Elmer, Boston, MA, USA) binding to intact cells in suspension was measured in 96-well plates with sodium phosphate buffer in triplicate as described in our previous work (Liang et al. 2009;Schmitt and Reith 2011). Increasing concentrations of non-radioactive CFT were included in the assay mixture to generate final CFT concentrations of 1,3,10, 30, 100 or 300 nM. Nonspecific binding was defined with 1 μM CFT. Biorad-DC protein kit was used to determine the cellular protein level.