Fig. 2: Capacity to phosphorylate rDAT at Thr53 is required for rKOR activation to elevate transporter surface expression.

EM4 cells were transfected with either WT DAT or DAT Ala53 plus rKOR, subjected to U69,593 and biotinylated as described in Methods. A Treatment with U69,593 did not affect total DAT band densities at ~55–60 kDa and ~85–90 kDa, derived from total protein lysates (n = 3 independent observations per condition, ~55–60 kDa and ~85–90 kDa bands were analyzed using two-way ANOVA, Šídák’s multiple comparisons test). B Incubation with U69,593 increased the WT DAT band densities at ~55–60 kDa and ~85–90 kDa, derived from biotinylated protein lysates. No effect of U69,593 was observed on the DAT Ala53 band densities (n = 3 independent observations per condition, ~55–60 kDa and ~85–90 kDa bands were analyzed using two-way ANOVA, Šídák’s multiple comparisons test). C, D Protein lysates were immunoblotted with p-Thr53 antibody. Bands marked with an asterisk indicate non-specific immunoreactivity. C In total protein lysates, incubation with U69,593 increased the density of the p-Thr53 immunoreactive band at ~85–90 kDa for WT DAT. No effect was observed on the band densities at ~55–60 kDa. No specific bands were detected for the DAT Ala53 variant (n = 4 independent observations per condition, WT DAT bands at ~55–60 kDa and ~85–90 kDa were analyzed using Student’s two-tailed unpaired t-test). D U69,593 augmented p-Thr53 immunoreactive band densities at ~55–60 and ~85–90 kDa for biotinylated WT DAT (n = 4 independent observations per condition, WT DAT bands at ~55–60 kDa and ~85–90 kDa were analyzed using Student’s two-tailed unpaired t-test). No specific bands were detected for the DAT Ala53 variant. All bars represent the mean with error bars reflecting the SD. Independent observations were performed in singlicate. Individual quantifications are displayed as individual symbols. *=P < 0.05; **=P < 0.01; n.s.=not significant; n.d. =not detected.