Agonist-induced internalization of OX1R in presence or down-regulation of Dynlt1/Dynlt3.

<p>Cell surface detection of OX1R was measured by ELISA. HEK293 cells were transfected with either 0.1 (A) or 0.25 µg (B, C, D) control pSG5-V5-His or pSG5-V5-His-OX1R, in combination with: (<b>A</b>) pCS2-Myc or pCS2-Myc-Dynlt1, (<b>B</b>) 10 nM of either control siRNA or <i>Dynlt1</i> siRNA (80% down-regulation), or (<b>C, D</b>) either 20 nM control siRNA or a combination of <i>Dynlt1</i> and <i>Dynlt3</i> siRNAs at 10 nM each (75% and 80% down-regulation, respectively). Cells were then treated with OX-A for up to 30 min. Normalized OD values refer to OD values that have been corrected (background-subtracted) and then expressed relative to basal conditions (% of value before stimulation for each condition). For all panels, there is maximum OX1R at the membrane at basal conditions (0 min; without OX-A) and the receptor is internalized (represented by loss at the membrane) after OX-A treatment. Results represent the mean ± SEM of 3 experiments, each performed in triplicate (except for panel D; results represent the mean ± SEM of 2 experiments). There is no statistical significance for the effect of over-expressing or down-regulating Dynlt1 and/or Dynlt3 as assessed by two-way ANOVA.