Figure 4. Y5R antagonist blocks NPY-induced Y5R-EGFP internalization.
A. A representative time-lapse fluorescence microscopy image of a cell transfected with Y5R-EGFP alone, with the marked region of membrane localization of the receptor that was subjected to analysis (white box). For this region, NPY-induced changes in receptor localization were measured by comparing ratios of fluorescence at the membrane to the corresponding sub-membrane cytosol region (membrane/cytosol) before and after treatment, as indicated by the numbers under the representative images. The linear graph represents the fluorescence densities across the cell membrane (along the yellow arrow) measured at the selected region at three time points. The bar graph depicts average membrane/cytosol ratios at the selected time points upon NPY treatment for all cells analyzed (11 independent regions). B. Analysis of Y5R-EGFP internalization upon treatment with 10−7M NPY in the presence of Y5R antagonist, CGP71683, at a concentration of 10−6M. The measurements and analyses were performed for 5 independent regions, as described for panel A. NT – non-treated cells; Y5A – cells upon Y5R antagonist pre-treatment; PM – plasma membrane. *** p<0.001 vs. NT by paired t-test.