esentative immunoprecipitation is provided for , , , and. For all bar graphs, values represent mean _ SEM of a minimum of four independent experiments. *p 0.05, **p 0.01, ***p 0.001. See also Figure S4. Perino et al. Page 14 Mol Cell. Author manuscript; available in PMC 2012 January 24. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 4. PKA Phosphorylates and Inhibits VX-222 HCV protease inhibitor p110γ Phosphorylation of p110γ immunoprecipitated from transfected HEK293T cells in the presence of recombinant PKA C, 32P-ATP, and PKA inhibitor PKI or vehicle. PKA-mediated phosphorylation of p110γ immunoprecipitated from HEK293T cells upon stimulation with forskolin. PKA-mediated phosphorylation of p110γ or p110γ K126A, R130A immunoprecipitated from HEK293T cells upon stimulation with forskolin.
Quantitative densitometry Geldanamycin HSP90 inhibitor of the experiment represented in. PKA-mediated phosphorylation of p110γ or p110γ T1024D mutant immunoprecipitated from HEK293T cells and incubated or not with active PKA. Quantitative densitometry of PKA phosphorylation of the experiment represented in. Lipid kinase activity of recombinant p110γ-GST incubated in vitro with or without recombinant PKA C. Lipid kinase activity of p110γ immunoprecipitated from transfected HEK293T cells treated with vehicle, pan-p110 inhibitor LY-294002 , forskolin or FSK plus PKA inhibitor Myr-PKI. Measurement of cellular PtdIns P3 levels in transfected HEK293T treated with the GPCR agonist PGE2 alone or in combination with FSK. Lipid kinase activity of p110γ wild-type or p110γ T1024D mutant immunoprecipitated from transfected HEK293T cells.
In lipid kinase assays , the ability of p110γ to phosphorylate phosphoinositide was detected by autoradiography following incubation with 32P-ATP substrate. A representative assay is presented in all figures. For all bar graphs, values represent mean _ SEM of a minimum of four independent experiments. *p 0.05, **p 0.01, ***p 0.001. See also Figures S5 and S6. Perino et al. Page 15 Mol Cell. Author manuscript; available in PMC 2012 January 24. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 5. PKA Inhibits the Lipid Kinase Activity of p110γ in the Myocardium Lipid kinase activity of p110γ immunoprecipitated from myocardial tissue lysates following treatment of wild-type or p110γ knockout mice with β-AR agonist isoproterenol or vehicle.
Measurement of myocardial PtdIns P3 levels in wild-type ,p110γ kinase-dead , and p110β kinase-dead mice treated with isoproterenol or vehicle. Phospho-Akt and total Akt levels in myocardial tissue lysates following treatment of wild-type , p110γ kinase-dead , and p110β kinase-dead mice with β-AR agonist isoproterenol or vehicle. Lipid kinase activity of p110γ immunoprecipitated from myocardial tissue lysates following ex vivo cardiac Langendorff perfusion with vehicle, isoproterenol , or ISO plus PKA inhibitor H89. Lipid kinase activity of p110γ immunoprecipitated from rat adult cardiomyocytes treated with isoproterenol , ISO plus PKA inhibitor Myr-PKI , or vehicle. Lipid kinase activity of p110γ immunoprecipitated from myocardial tissue lysates of mice subjected to transverse aortic constriction for 1 week or to sham operation.
Insets are representative hematoxylin and eosin stainings of left ventricular sections from sham-operated and 1 week TAC mice. A representative assay is presented in all figures. For all bar graphs, values, obtained by quantitative densitometry and normalized over control, represent mean _ SEM of a minimum of five mice per group. *p 0.05, **p 0.01. Perino et al. Page 16 Mol Cell. A