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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 4.

Figure 4

Gαs but not PKA is critical for β2AR-mediated ERK phosphorylation. A) IF showing GFP expression of FLAG-β2AR Gnas f/f MEFs transduced with control adenoviral (adeno)-GFP or adeno-Cre-GFP, and western blot of pERK upon 3 min Iso stimulation of these cells (representative of three independent experiments). B) Western blot for Gαs in 293 CRISPR/Cas9-edited Gαs KO cells. C) Western blot of pERK upon 3 min Iso or EGF (10 ng/mL) stimulation in the indicated cells. Representative of three independent experiments. (D and E) Relative amounts (mean +/- SEM of three independent experiments) of (D) cAMP and (E) CRE luciferase activity in the indicated cells upon Iso stimulation. F) Western blot of pERK after Iso stimulation in Gαs KO FLAG-β2AR cells transfected with vector control or Gαs. Representative of three independent experiments. (G and H) Western blot of pERK in 293 FLAG-β2AR cells stimulated with Iso after pretreatment with DMSO (control), (G) H89 (10 μM), or (H) cAMPS-RP (100 μM). Representative of three independent experiments for G and H. I) Relative CRE luciferase activity in Iso and FSK (5 mg/ml) stimulated 293 FLAG-β2AR cells pretreated with H89, cAMPS-RP or DMSO control; mean +/- SEM of three experiments. J) IF of GFP expression, relative CRE luciferase activity (mean +/- SEM, three experiments), and western blot of pERK in 293 FLAG-β2AR cells transfected with GFP-PKI or GFP-PKI-mutant (PKI-Mut) plasmids upon stimulation with Iso (representative of three independent experiments).