goat polyclonal anti dat  (Santa Cruz Biotechnology)

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: [ 3 H]Dopamine Uptake through the Dopamine and Norepinephrine Transporters is Decreased in the Prefrontal Cortex of Transgenic Mice Expressing HIV-1 Transactivator of Transcription Protein

doi: 10.1124/jpet.120.266023

Figure Lengend Snippet: Kinetic analysis of synaptosomal [3H]DA uptake was determined in the PFC of iTat-tg mice after 7 or 14 days’ administration of saline or Dox. Synaptosomes were incubated with a range of mixed DA concentrations (0.1–5 µM, final concentration) containing a fixed concentration (12 nM) of [3H]DA. The Vmax and Km values for [3H]DA uptake via DAT (A and C) or NET (B and D) in the PFC of iTat-tg (iTat) mice after 7 (A and B) or 14 (C and D) days’ administration of saline or Dox were calculated using nonlinear regression analysis with a one-site binding parameter and represent the means from five to seven independent experiments ± S.E.M. *P < 0.05; **P < 0.01 compared with saline control group. SAL, saline.

Article Snippet: Samples were separated by 10% SDS-polyacrylamide gel electrophoresis for ∼90 minutes at 125 V. Samples were then transferred to Immobilon-P transfer membranes (0.45-µm pore size; Millipore Co., Bedford, MA) in transfer buffer (50 mM Tris, 250 mM glycine, 3.5 mM SDS) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA) for 90 minutes at 75 V. The membranes were then incubated with blocking buffer (5% milk powder in PBS containing 0.5% Tween-20) for 1 hour at room temperature, followed by incubation with either goat anti-DAT (C-20 polyclonal antibody, diluted 1:500 in blocking buffer; Santa Cruz) or mouse anti-NET (MAb tech, 05-1 monoclonal antibody, diluted 1:5000 in blocking buffer) overnight at 4°C.

Techniques: Incubation, Concentration Assay, Binding Assay

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: [ 3 H]Dopamine Uptake through the Dopamine and Norepinephrine Transporters is Decreased in the Prefrontal Cortex of Transgenic Mice Expressing HIV-1 Transactivator of Transcription Protein

doi: 10.1124/jpet.120.266023

Figure Lengend Snippet: Analysis of plasmalemmal surface expression of DAT and NET was determined in the PFC of iTat-tg mice after 7 days’ administration of saline or Dox. Synaptosomes were incubated with sulfo-NHS-biotin and Pierce monomeric avidin beads and washed multiple times to isolate the DAT or NET, which were present on the plasmalemmal membrane. Top panels: representative immunoblots of total and biotinylated (Biotin) fraction of DAT (A) or NET (B) from the PFC of iTat-tg (iTat) mice from Dox-treated and saline-treated (SAL) control groups. Calnexin was used as a control protein. Bottom panels: the ratio of total or biotinylated DAT (A) or NET (B) immunoreactivity to calnexin immunoreactivity expressed as means ± S.E.M. from five independent experiments.

Article Snippet: Samples were separated by 10% SDS-polyacrylamide gel electrophoresis for ∼90 minutes at 125 V. Samples were then transferred to Immobilon-P transfer membranes (0.45-µm pore size; Millipore Co., Bedford, MA) in transfer buffer (50 mM Tris, 250 mM glycine, 3.5 mM SDS) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA) for 90 minutes at 75 V. The membranes were then incubated with blocking buffer (5% milk powder in PBS containing 0.5% Tween-20) for 1 hour at room temperature, followed by incubation with either goat anti-DAT (C-20 polyclonal antibody, diluted 1:500 in blocking buffer; Santa Cruz) or mouse anti-NET (MAb tech, 05-1 monoclonal antibody, diluted 1:5000 in blocking buffer) overnight at 4°C.

Techniques: Expressing, Incubation, Avidin-Biotin Assay, Western Blot

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: [ 3 H]Dopamine Uptake through the Dopamine and Norepinephrine Transporters is Decreased in the Prefrontal Cortex of Transgenic Mice Expressing HIV-1 Transactivator of Transcription Protein

doi: 10.1124/jpet.120.266023

Figure Lengend Snippet: Saturation binding of [3H]WIN 35,428 or [3H]nisoxetine in the PFC of iTat-tg mice after 7 days’ administration of saline (SAL) or Dox (DOX). For [3H]WIN 35,428 binding to DAT, synaptosomes were incubated in assay buffer with one of six concentrations of [3H]WIN 35,428 (1–30 nM, final concentration) and 1 µM desipramine on ice for 2 hours. Nonspecific binding was determined in the presence of 10 µM cocaine. For [3H]nisoxetine binding to NET, synaptosomes were incubated in assay buffer with one of six concentrations of nisoxetine (0.5–30 nM, final concentration) along with a fixed concentration of [3H]nisoxetine (3 nM) and 0.1 µM GBR12909 on ice for 2 hours. Nonspecific binding was determined in the presence of 10 µM desipramine. The Bmax and Kd values for [3H]WIN 35,428 (A) or [3H]nisoxetine (B) binding to DAT or NET in the PFC of iTat-tg (iTat) mice after 7 days’ administration of saline or Dox were calculated using nonlinear regression analysis with a one-site binding parameter and represent the means ± S.E.M. from five independent experiments. *P < 0.05; **P < 0.01 compared with saline control group. DA turnover rate values were determined for DAT from (C) the Vmax of [3H]DA uptake (Fig. 1A)/Bmax of [3H]WIN 35,428 binding and (D) the Km/Kd of [3H]DA uptake (Fig. 1A)/Bmax of [3H]WIN 35,428 binding and for NET from (E) the Vmax of [3H]DA uptake (Fig. 1B)/Bmax of [3H]nisoxetine binding and (F) the Km/Kd of [3H]DA uptake (Fig. 1B)/Bmax of [3H]nisoxetine binding.

Article Snippet: Samples were separated by 10% SDS-polyacrylamide gel electrophoresis for ∼90 minutes at 125 V. Samples were then transferred to Immobilon-P transfer membranes (0.45-µm pore size; Millipore Co., Bedford, MA) in transfer buffer (50 mM Tris, 250 mM glycine, 3.5 mM SDS) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA) for 90 minutes at 75 V. The membranes were then incubated with blocking buffer (5% milk powder in PBS containing 0.5% Tween-20) for 1 hour at room temperature, followed by incubation with either goat anti-DAT (C-20 polyclonal antibody, diluted 1:500 in blocking buffer; Santa Cruz) or mouse anti-NET (MAb tech, 05-1 monoclonal antibody, diluted 1:5000 in blocking buffer) overnight at 4°C.

Techniques: Binding Assay, Incubation, Concentration Assay