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. 2015 Feb 27;290(17):10814–10827. doi: 10.1074/jbc.M114.612192

FIGURE 6.

FIGURE 6.

In vivo TAT-NET-Thr30 peptide administration attenuates NET up-regulation by cocaine. Mice were injected with vehicle (Veh), TAT-NET-Thr30, or TAT-NET-T30A (12 mg/kg intravenously via the tail) 15 min prior to saline (Sal) or cocaine (Coc) (30 mg/kg intraperitoneally) injections. Brains were collected 1 h postinjection for PFC synaptosomal preparations. A, NE uptake. NE uptake was measured as described under “Experimental Procedures.” 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(4,20) = 13.36 for TAT-NET-Thr30 and F(4,20) = 15.31 for TAT-NET-T30A, p < 0.01). B, NET surface biotinylation. PFC synaptosomes obtained from drug-administered mice 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) = 31.67 for TAT-NET-Thr30 and F(4,8) = 23.00 for TAT-NET-T30A, p < 0.05). TH immunoblots corresponding to total are shown for equal protein loading. C, DAT-mediated DA uptake. DAT-mediated DA uptake was measured as described under “Experimental Procedures.” Uptake data derived from three separate experiments, each in triplicate, are given as mean ± S.E. (error bars). * indicates a significant change in DA transport (one-way analysis of variance; Dunnett's test: F(4,28) = 12.65, p < 0.01). D, DAT surface biotinylation. DATs from NAc synaptosomes obtained from drug-administered mice were biotinylated as described above for NET, and avidin-bound (B), total (T), and unbound (UB) fractions were subjected to SDS-PAGE and immunoblotting with DAT antibody. Representative blots show a DAT-specific band at ∼75 kDa. Mean values of quantified surface DAT band densities (normalized to TH levels in the total blot) from two separate experiments are given in the bar graph below.