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. 2020 Mar 4;295(16):5229–5244. doi: 10.1074/jbc.RA120.012628

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© 2020 Fagan et al.

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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Figure 8. — The DAT N terminus is required for PKC-stimulated internalization. DAT internalization assays. WT DAT and DAT/SERT chimera internalization rates were measured with or without 1 μm PMA or with or without 20 μm AIM-100 for 10 min at 37 °C, as described under “Experimental procedures.” Top panels, representative immunoblots showing total surface expression at t = 0 (T), strip control (S), and internalized protein during either vehicle (V), PMA (P), or AIM-100 (A) treatments. Bottom panels, averaged data. A, stimulated internalization in stable SK-N-MC cell lines. Rates are expressed as percentage of vehicle-treated controls ± S.E. (error bars). Asterisks, significant difference from vehicle controls (one-way ANOVA with Dunnett's multiple-comparison test, n = 8–13; DAT: ANOVA F_{(2, 34)} = 7.94, p = 0.0015; vehicle versus PMA: **, p = 0.007; vehicle versus AIM-100: **, p = 0.002; N-S/DAT: ANOVA F_{(2, 21)} = 4.38, p = 0.03; vehicle versus PMA: p = 0.82; vehicle versus AIM-100: *, p = 0.02; DAT/C-S: ANOVA F_{(2, 30)} = 5.22, p = 0.01; vehicle versus PMA: *, p = 0.01; vehicle versus AIM-100: **, p = 0.026. S/DAT/S: ANOVA F_{(2, 21)} = 0.84, p = 0.44). B, basal internalization in stable SK-N-MC cell lines. Chimera basal internalization rates did not significantly differ from WT DAT (one-way ANOVA F_{(3, 39)} = 4.046, p = 0.013; Dunnett's multiple-comparison test: DAT versus N-S/DAT: p = 0.54; versus DAT/C-S: p = 0.06; versus S/DAT/S: p = 0.72, n = 9–12). C, DAT and N/S-DAT internalization in stable SK-N-DZ cell lines. Average internalization rates are expressed as percentage of surface protein internalized/10 min ± S.E. Asterisks, significant difference from the indicated protein or treatment (two-way ANOVA: interaction: F_{(1, 16)} = 1.06, p = 0.32; construct: F_{(1, 16)} = 15.6, p = 0.001; drug: F_{(1, 16)} = 25.6, p = 0.0001; Tukey's multiple-comparison test: DAT(veh) versus DAT(PMA): *, p = 0.01, DAT(veh) versus N-S/DAT(veh): *, p = 0.03; N-S/DAT(veh) versus N-S/DAT(PMA): p = 0.21, n = 4–7).