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. Author manuscript; available in PMC: 2018 Jun 20.
Published in final edited form as: Sci Signal. 2017 Jun 20;10(484):eaal3395. doi: 10.1126/scisignal.aal3395

Figure 5.

Figure 5

β2AR-mediates ERK activation through Gβγ signaling and activation of a signaling cascade that involves SRC, SHC, SOS, RAS, RAF, and MEK. (A and B) Western blot of pERK after Iso or EGF stimulation (3 min) in 293 FLAG-β2AR cells pretreated with DMSO or (A) U0126 (10 μM), (B) SB-590885 (SB, 10 μM) or GDC-0879 (GDC, 10 μM). Representative of three independent experiments for A and B. C) Western blot of pERK after Iso stimulation in 293 FLAG-β2AR cells transfected with DN K-RAS, DN H-RAS or control. Representative of four independent experiments. D) Relative CRE luciferase activity in Iso and FSK-stimulated 293 FLAG-β2AR cells transfected with DN K-RAS or control plasmid; mean +/- SEM of three experiments. E) Western blot showing active RAS-GTP pull down (IP) and input for RAS and α-tubulin after Iso stimulation. Representative of three independent experiments. F) IF showing GFP expression (scale bar, X) and pERK western blot of Iso-stimulated FLAG-β2AR-Rasless MEFs transduced with adeno-GFP or adeno-Cre-GFP virus (representative of three independent experiments). G) Effects of siRNA library screen of various RAS-GEFs on Iso-stimulated pERK amounts. Representative of three independent experiments. H) Western blot of pERK in 293 FLAG-β2AR cells transfected with siRNA to SOS1 and SOS2 (SOS) or control in response to Iso. Representative of three independent experiments. (I and J) Western blot of pSrc and pSHC in (I) response to Iso stimulation, and (J) with SRC inhibitors PP1 (10 μM), SU6656 (10 μM), or DMSO. Representative of three independent experiments for I and J. K) Western blot of pERK in 293 FLAG-β2AR cells pretreated with SRC inhibitors upon Iso stimulation. Representative of three independent experiments. L) Western blot of pSRC in the indicated cells upon Iso stimulation. Representative of three independent experiments. M) Western blot of pERK after Iso or EGF stimulation in 293 FLAG-β2AR cells transfected with control or HA-tagged GRK2ct plasmids. Representative of three independent experiments. N) Western blot of Iso-stimulated pSRC and pERK in 293 FLAG-β2AR cells transfected with control or HA-tagged GRK2ct. Representative of three independent experiments.