FIGURE 5.
In vitro treatment of mouse PFC synaptosomes with TAT-NET-Thr30 peptide blocks cocaine-induced NET up-regulation and phosphorylation. Mouse PFC synaptosomes were preincubated with vehicle (Veh) or 10 μm TAT-NET-Thr30 or TAT-NET-T30A at 37 °C for 15 min. Incubations were continued in the presence or absence of 50 μm cocaine (Coc) for 60 min. A, NE uptake. Data derived from three separate experiments, each in triplicate, are given as mean ± S.E. (error bars). * indicates a significant change in NE transport (one-way analysis of variance; Dunnett's test: F(4,32) = 6.16, p < 0.05). B, NET surface biotinylation. Synaptosomes treated as above were biotinylated, and biotinylated NETs were isolated by avidin binding. Equal aliquots from total (T) and avidin-unbound fractions (UB) and entire eluates from avidin beads representing bound fractions (B) were loaded onto gels, and the blots were probed with commercially available mouse NET monoclonal antibody as described under “Experimental Procedures.” Representative blots show a NET-specific band at ∼85 kDa. Quantified surface NET densities (normalized to TH levels in the total blot) from three separate experiments are given in bar graphs as mean ± S.E. (error bars). * indicates a significant change from vehicle control (one-way analysis of variance; Dunnett's test: F(4,8) = 4.21, p < 0.05). TH immunoblots corresponding to total are shown for equal protein loading. C, NET phosphorylation. Following metabolic labeling with 32P, synaptosomes were treated with drugs as described above and solubilized. The synaptosomal extract was subjected to immunoprecipitation with NET-82 polyclonal antibody and autoradiography as described as described under “Experimental Procedures.” A representative autoradiogram (Autorad) from two independent experiments shows phospho-NET (p-NET) bands and enhanced density of phospho-NET following cocaine treatment compared with vehicle, TAT-NET-Thr30, or TAT-NET-Thr30 + cocaine. Parallel immunoprecipitations using unlabeled synaptosomes show pulldown of an equal amount of NET following drug treatments (shown in the immunoblot below the autoradiogram). Mean values of quantified phospho-NET density (normalized to NET pulldowns from unlabeled synaptosomes) from two experiments are given below in the bar graph.