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. Author manuscript; available in PMC: 2014 Jul 23.
Published in final edited form as: Circulation. 2013 Jun 19;128(4):10.1161/CIRCULATIONAHA.113.003183. doi: 10.1161/CIRCULATIONAHA.113.003183

Figure 3.

Figure 3

βAR desensitization by TNFα is GRK2 dependent. (A) Immunoblots to assess phospho-β2AR, GRK2 and FLAG-β2AR on the plasma membranes of TNFα and ISO treated HEK-FLAG-β2AR cells, (n=4). (B) Levels of β2AR phosphorylation and GRK2 co-immunoprecipitating with FLAG-β2AR from plasma membrane fractions of HEK-FLAG-β2AR cells following TNFα or ISO, (n=4). (C) Representative autoradiograph showing β2AR phosphorylation upon TNFα or ISO treatment following metabolic [32]Pi labeling of HEK-FLAG-β2AR cells (n=4). (D) HEK 293 cells were transfected with FLAG-β2AR Wt or Serine 355/356 mutant cDNA constructs, metabolically labeled with [32]Pi, treated with TNFα. Representative autoradiograph showing β2AR phosphorylation following immunoprecipitation with anti-FLAG antibody (n=4). (E) Effect of GRK2 knock down by siRNA on phosphorylation of β2ARs following the stimulation of HEK-FLAG-β2AR cells with ISO or TNFα. (F) Cumulative data showing significant loss of β2AR phosphorylation due to knock down of GRK2 by siRNA (right panel), (n=3–4), *p< 0.001 versus Vehicle (Veh), # p<0.005 versus ctrl siRNA -TNFα or ISO.