Fig. 8.

Effect of SHIP2 inhibition on DAT activity. a HT22 cells stably transfected with BirA*-DAT were preincubated for 30 min in the presence of the indicated concentrations of the SHIP2 inhibitor AS19494490. Transport was determined in the presence of 0.01 µM [³H]-DA for 10 min. Values correspond to the mean ± ESM of three triplicate determinations and are presented as a percentage of the untreated controls. b SH-SY5Y cells were incubated for the indicated times in the presence of AS1949490 (10 µM) and [³H]-DA; uptake was measured and data are presented as in a. c SH-SY5Y cells were incubated for 10 min in the presence of AS1949490 (10 µM) and then the compound was washed out for the indicated times; [³H]-DA transport was measured for 10 min as in a. d Synaptosomes isolated from rat striatum were preincubated for 10 min the presence of AS1949490 (10 µM) and then incubated in the presence of 0.01 µM [³H]-DA for 10 min (***p < 0.001, unpaired t test). e [³H]-DA uptake kinetics in SH-SY5Y cells in the absence (○) or presence of AS1949490 (10 µM, 20 min) (●). Values correspond to the mean ± SEM of two quadruplicate determinations. f HEK293 cells were transfected mCherry-DAT and 2 days later they were incubated in the absence or presence of AS1949490 (10 µM, 20 min). Cells were subjected to NHS–SS–biotinylation as described in Materials and Methods. Samples corresponding to biotinylated protein isolated with streptavidin–agarose beads (Biot), or non-biotinylated protein remaining after streptavidin–agarose precipitation (N Biot), or total protein in the lysates (Lys) were electrophoresed and immunoblotted,. Immunoblots were probed with anti-mCherry and the bands were visualized using the ECL method. Histograms represent the quantification of the intensities the biotinylated protein and are the average ± SEM of three experiments