Figure 1.

ALs enhance DAT oligomerization. A, structure of AIM-100, AL3, AL4, AL8, and AL9. Central scaffolds are indicated by red punctuate lines. See Fig. S1 for the full list, names, and structures of all tested compounds. B, PAE/YFP-HA-DAT cells were incubated with vehicle (Veh; DMSO), AIM-100, AL3, AL4, AL8, or AL9 (all 20 μM) at 37 °C for 2 h. Lysates were resolved by SDS-PAGE and probed by Western blotting with the GFP antibody. Mean values (±SD) of the fraction of trimers [T-DAT] of total mature YFP-HA-DAT (M-DAT+T-DAT) immunoreactivity are shown below the GFP blot (n = 3). C, PAE/YFP-HA-DAT cells were untreated or incubated with vehicle (Veh; DMSO) or AL3 (20 μM) at 37 °C for 1 h, washed with KRHG and incubated with 4 mM BS³ for 15 min at 37 °C in KRHG. Lysates were resolved by SDS-PAGE and probed by western blotting with the GFP antibody. Mean values (±SD) of the fraction of trimers [T+hO] of total mature YFP-HA-DAT immunoreactivity are shown below the GFP blot (n = 3). D, striatal synaptosomes prepared from HA-DAT mice were incubated with vehicle, AIM-100, AL3, AL4, AL9 (all 20 μM) or AL8 (20–40 μM) in KRHG (left panel) or KRHG/F12 (1:1) (right panel) at 37 °C for 2 h. Lysates were resolved by electrophoresis and probed by Western blotting with the DAT antibody. Mean values (±SD) of the fraction of trimers and high-order oligomers (T+hO) of total HA-DAT immunoreactivity were calculated from the data obtained in three mice that were treated in independent experiments (n = 3). AL, AIM-100–like; DAT, dopamine transporter; DMSO, dimethyl sulfoxide; hO, high order oligomers; im-M, immature monomers; im-T, immature trimers; KRHG, glucose-supplemented Krebs-Ring buffer; M, monomers; PAE, porcine aortic endothelial; T, trimers; YFP-HA-DAT, YFP- and HA-epitope tagged DAT.