Figure 2
From: Probing the stoichiometry of β2-adrenergic receptor phosphorylation by targeted mass spectrometry
β2AR phosphorylation in HEK293 cells challenged with beta-adrenergic agonist, forskolin, carazolol or ICI118,551. HEK293 cells stably expressing N-terminal Flag-tagged, C-terminal eGFP-tagged β2AR treated without (NT) or with either 10 μM isoproterenol (Iso), 10 μM forskolin (FSK), 10 μM carazolol (Crz) or 10 μM ICI118,551 (ICI) for 5 or 10 min. β2AR was immunoprecipitated with anti-Flag immunoadsorption beads. SDS-PAGE and immunoblotting were performed as indicated in the Materials and methods section. The phosphorylation of β2AR was normalized to total β2AR. The data were quantified and are displayed as percentage of phosphorylation of each site (s) sampled at 5-min following stimulation with the beta-adrenergic agonist isoproterenol. The experimental data shown are of a single analysis performed in triplicate and replicated multiple times with similar results. (A) Quantification of phosphorylation of S262, S345/346 and S355/356. (B) Representative westernblotting image.