Dual-localization of co-expressed β2-adrenergic receptors after antagonist-mediated recycling. HEK293 cells stably expressing Flag-β2AR-Gαs receptor were transiently transfected with HA-β2AR construct. After 48 hours, receptor internalization was induced by incubating cells with 1 μM isoproterenol for 5 hours. 1 μM propranolol was added (Agonist + Antagonist) or not (Agonist) for a further 60 minutes to stop receptor endocytosis. Fixed and immuno-stained cells were subjected to epifluorescence microscopy, as described in Methods, to visualize the sub-cellular localization of HA-β2AR (Alexa-Fluor 594 staining; red) and Flag-β2AR-Gαs (Alexa-Fluor 488 staining; green). Scale bars, 10 μm. Note that the antagonist allows the membrane return of the wild type receptor but not of the chimeric protein. Photographs represent the typical situation observed in 4 independent experiments.
Di Certo et al. BMC Cell Biology 2008 9:56 doi:10.1186/1471-2121-9-56