Overview of hNET-Tat complex structure.

(A) hNET and Tat are showed in cyan and gold surface style, respectively. (B) Local view of the favorable hydrogen bond between the positively charged N-terminal amino group of residue M1 in Tat and the hydroxyl group of residue Y467 in hNET with labeled distance.</p

Inhibitory affinity of substrates and inhibitors for [³H]DA and [³H]NE uptake in WT hNET and its mutants.

Inhibitory affinity of substrates and inhibitors for [3H]DA and [3H]NE uptake in WT hNET and its mutants.</p

S1 File -

(DOCX)</p

Effects of Tat_1-86 on the specific [³H]DA uptake in WT-, Y467H-, and Y467F-hNET.

PC12 cells transfected with the WT hNET or mutants were preincubated with or without recombinant Tat1-86 (rTat1-86, 140 nM, final concentration) at room temperature for 20 min followed by the addition of [3H]DA (50 nM, final concentration) for an additional 8 min at room temperature. In parallel, nonspecific uptake was determined in the presence of 10 μM desipramine. Data are expressed as means ± S.E.M. from 4–9 independent experiments. * p t test).</p

Kinetic properties of the reuptake of [³H]DA and [³H]NE in PC12 cells expressing WT hNET and its mutants.

Kinetic properties of the reuptake of [3H]DA and [3H]NE in PC12 cells expressing WT hNET and its mutants.</p

Mutational effect of hNET Tyr467 residue on functional efflux of DA or MPP⁺.

CHO cells transfected with WT hNET or mutants were preincubated with assay buffer containing 50 nM [3H]DA for 20 min or 5 nM [3H]MPP+ for 30 min at room temperature. After incubation, cells were washed and incubated with fresh buffer at indicated time points. Subsequently, the buffer was removed, and radioactivity in the buffer and residual radioactivity in the cells was counted. Efflux fractions of [3H]DA or [3H]MPP+ in WT hNET or mutants were collected every 10 mins from 0 time point until 110 mins. Fractional efflux of [3H]DA or [3H]MPP+ are expressed as a percentage of baseline efflux at 0 time point at the start of the experiment. (A) [3H]DA efflux plotted as a percentage of baseline release after 20 minute preloading period. (B) [3H]MPP+ efflux plotted as a percentage of baseline release after 30-minute preloading period.</p

Kinetic properties of [³H] WIN35,428 and [³H] Nisoxetine binding in PC12 cells expressing WT hNET and its mutants.

Kinetic properties of [3H] WIN35,428 and [3H] Nisoxetine binding in PC12 cells expressing WT hNET and its mutants.</p

Cell surface expression of WThNET, Y467F, and Y467H was determined by biotinylation and Western blotting.

(A) Representative immunoreactive blots for NET and calnexin (as control protein) in total, non-biotinylated (intracellular) and biotinylated (surface expression) fractions. (B) The quantification of total, non-biotinylated (INTRA) and biotinylated (BIOTIN) expression of NET was expressed as mean ± S.E.M of the ratio of total, non-biotinylated or biotinylated NET immunoreactivity to Calnexin immunoreactivity from 5 independent experiments. Raw immunoblots are provided in S1 File.</p