β2AR-Gαs and β2AR co-localization with endocytosed transferrin. A. The cells were incubated for 30 min with 100 μM Alexa-Fluor 488 conjugated transferrin, a marker of early and recycling endosomes, and with 1 μM isoproterenol for another 30 minutes. The samples were then washed, fixed and immuno-stained with antibody against Flag-epitopes, as described in Methods. The sub-cellular distribution and co-localization of both receptors with transferrin were revealed by confocal microscopy. The data are representative images from three independent experiments. Red, Flag-tagged receptor Alexa-Fluor 594 stained; Green, Alexa-Fluor 488 conjugated transferrin. In the merged images, numerous yellow vesicular spots, corresponding to the co-localization between internalized receptors and transferrin, are visualized. Scale bars, 10 μm. B. The graph indicates the fraction of endocytic vesicles in which internalized receptors co-localized with the Alexa-Fluor 488 conjugated transferrin. Error bars represents the S.D. of data collected from multiple fields (n = 6) from 4 independent experiments.
Di Certo et al. BMC Cell Biology 2008 9:56 doi:10.1186/1471-2121-9-56