Dynlt1 alters the intracellular localization of OX1R following ligand-induced internalization.

<p>(<b>A</b>) HEK293 cells were transfected with pEGFP-N1-OX1R and stimulated with OX-A for the indicated times. IB analysis of whole cell lysates shows that ERK1/2 phosphorylation can be achieved after stimulation of this OX1R-GFP fusion protein with OX-A. (<b>B</b>) HEK293 cells were transfected with pCS2-Myc-Dynlt1 and pEGFP-N1-OX1R or corresponding empty vector. Whole-cell lysates were subjected to IP with an anti-GFP antibody to immunoprecipitate OX1R-GFP and anti-Myc antibody to reveal Myc-Dynlt1. Equal transfection of Myc-Dynlt1 was verified by IB with anti-Myc antibody on cell extracts (input). (<b>C, D</b>) HEK293 cells stably expressing OX1R-GFP were transfected with control plasmid (<b>C</b>) or pCS2-FLAG-Dynlt1 (<b>D</b>), stimulated with OX-A for 5–30 min or left un-stimulated (0 min) and analyzed by fluorescent microscopy. Cells were labeled with an antibody specific for a marker of early endosomes, EEA1. Maximum co-localization of green pixels with red pixels occurs after 15 min OX-A in control conditions. However, when over-expressing Dynlt1, co-localization is rather similar across OX-A stimulation times. Left panels: green GFP fluorescence; OX1R, middle panels: EEA1 labeling, red ALEXA 568 secondary antibody; early endosomes, and right panels: the merged images. (<b>E</b>) Magnification of merged fields from panels C and D (white squares) after 15 min OX-A without or with Dynlt1 over-expression. White arrows indicate OX1R-GFP signal (green) not co-localized with endosome marker (red). (<b>F</b>) Manders' co-localization coefficient for green channel (OX1R-GFP) was generated on images that were co-localized using Openlab software. Manders' co-localization coefficient (OX1R): co-localized green and red pixels divided by total number of green pixels over threshold. Results represent the mean ± SEM of triplicates from one experiment, where at least 5 fields with 1–3 cells in each were analyzed for each time point. This experiment was done twice with similar results (three times for the 0 and 15 min time points). The co-localization of OX1R-GFP with the EEA1 marker for endosomes, after 15 min OX-A, is decreased upon Dynlt1 over-expression. *: p<0.05 when compared to control transfection (without Dynlt1) after 15 min OX-A.