Skip to main content
. 2021 Feb 18;296:100430. doi: 10.1016/j.jbc.2021.100430

Figure 8.

Figure 8

DAT mutant lacking amino-terminal residues 1-65 is oligomerized and endocytosed upon treatment with ALs.A, PAE/YFP-HA-DAT and PAE/YFP-HA-ΔN-DAT cells were incubated with vehicle, AIM-100, AL3, AL4, or AL8 (all 20 μM) for 2 h at 37 °C, and lysates were resolved by SDS-PAGE and immunoblotting with the GFP antibody. Representative experiment is shown. B, quantification of the fraction of trimeric species (T-DAT) relative to the total YFP-HA-DAT or YFP-HA-ΔN-DAT immunoreactivity in experiments exemplified in (A). Bar graph represents mean values of the mature T-DAT fraction of total mature YFP-HA-DAT or YFP-HA-ΔN-DAT (±S.D.; n = 3–4). Differences between the T fractions in YFP-HA-DAT and YFP-HA-ΔN-DAT treated with the same compound were not significant (p > 0.05). The p values for WT and mutant DATs were calculated for “AIM-100/ALs” versus corresponding “Veh” using multiple comparisons, paired, one-way ANOVA. C, PAE/YFP-HA-DAT and PAE/YFP-HA-ΔN-DAT cells were incubated with HA11 for 30 min at 37 °C and then incubated with vehicle (DMSO), AIM-100, AL3, AL4, or AL8 (all 20 μM) for 2 h at 37 °C. After fixation, cells were stained with secondary Cy5-conjugated anti-mouse antibodies (surface HA-DAT), permeabilized with Triton X-100, and stained with secondary Cy3-conjugated anti-mouse (internalized HA-DAT). 3D images were acquired through 488 (YFP, not shown), 561 (Cy3, red), and 640 nm (Cy5, cyan) channels. Maximum intensity projections of 3D images are presented. Scale bars, 10 μm. D, Cy3/Cy5 ratios were calculated in experiments exemplified in (C). Results are presented as mean values of the ratio (±SD). Differences in the ratio values between “YFP-HA-DAT” (wtDAT) and YFP-HA-ΔN-DAT (ΔN-DAT) treated with the same compound were not statistically significant (p > 0.05). The p values for WT and mutant DATs were calculated for “AIM-100/ALs” versus corresponding “Veh” using multiple comparisons, paired, one-way ANOVA. AL, AIM-100–like; DAT, dopamine transporter; DMSO, dimethyl sulfoxide; im-T-DAT, immature trimers; im-M-DAT, immature monomers; M-DAT, monomers; PAE, porcine aortic endothelial; T-DAT, trimers; YFP-HA-DAT, YFP- and HA-epitope tagged DAT.