AZD6482 1173900-33-8 cells transfected with p110γand PDE3B-Flag.

93T cells transfected with p110γand PDE3B-Flag. Coimmunoprecipitation of p110γ with PDE3B in wild-type but not in p110γknockout mouse neonatal cardiomyocytes. Phosphodiesterase activity in PDE3B immunoprecipitates upon transfection of HEK293T cells with PDE3B-Flag or with PDE3B-Flag AZD6482 1173900-33-8 and p110γ. Cells were treated with PKA inhibitor H89 or vehicle as indicated. PDE activity was calculated relative to the activity of single PDE3B transfectants. Phosphodiesterase activity of double p110γ, PDE3B-Flag transfectants treated with PKA inhibitor Myr-PKI or vehicle. PKA activity in a p110γ immunoprecipitate from transfected HEK293T cells. Coimmunoprecipitation of transfected p110γ, PDE3B-Flag, PKA RIIα-ECFP , and PKA CAT-YFP from HEK293T extracts. Coimmunoprecipitation of p110γ and p84/p87, but not p101, with PKA RIIα from HEK293T transfected cells.
Colocalization of p110γ and PKA RIIα by immunofluorescence in mouse adult cardiomyocytes. SB939 929016-96-6 Longitudinal and transverse sections are shown in the upper and right panels, respectively. Single p110γ and PKA RIIαlocalizations are presented in the lower panels. PDE3B, p110γ, p84/p87, and PKA CAT , but not p101, coimmunoprecipitate with PKA RIIα in myocardial tissue extracts of wild-type mice. A representative immunoprecipitation is presented in �? 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 Figures S1 and S2. Perino et al. Page 12 Mol Cell. Author manuscript; available in PMC 2012 January 24. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 2.
p110γ Is a Bona Fide AKAP In vitro copurification of p110γ-GST and PKA RIIα-6His in a GST pull-down assay. Direct interaction of p110γ and PKA RIIα in a surface plasmon resonance assay. p110γwas immobilized on the chip, and PKA RIIα was injected at three different concentrations. Binding of 32P-labeled PKA RIIα to immunoprecipitated p110γ in the presence of AKAP-IS scrambled peptide but not in the presence of AKAP-IS peptide. Competition of the p110γ-PKA RIIα coimmunoprecipitation with AKAP-IS peptide. Quantitative densitometry of the competition experiment represented in. Values represent mean _ SEM of four independent experiments. *p 0.05. Loss of the coimmunoprecipitation of PKA RIIα with p110γ by truncation of the 1�?5 amino acids of RIIα-ECFP.
A representative assay is presented in all figures. See also Figure S3. Perino et al. Page 13 Mol Cell. Author manuscript; available in PMC 2012 January 24. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 3. Mapping of the p110γ-PKA RIIα Interaction Loss of the coimmunoprecipitation of p110γ-Myc with PKA RIIα by deletion of amino acids 114�?80 but not by deletion of the Ras-binding domain or the PIK domain of p110γ-Myc. Sequence of the 126�?50 peptide is represented on a scheme of p110γ. In dosedependent competition experiments, the 126�?50 peptide, but not a scrambled control peptide, antagonized the p110γ-RIIα protein-protein interaction. Quantitative densitometry of the competition experiment represented in.
Binding of PKA RIIα to a set of alanine mutant 126�?50 p110γ peptides in a solid-phase peptide array. Mutation of K126 and R130 of p110γ to A blunts coimmunoprecipitation of p110γ with PKA RIIα in transfected HEK293T cells. Values were obtained by quantitative densitometry and normalized over control. Phosphodiesterase activity of PDE3B immunoprecipitates upon transfection of HEK293T cells with PDE3B-Flag alone or with PDE3B-Flag and either wild-type or mutant p110γ. A repr