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. Author manuscript; available in PMC: 2015 Jan 1.

Published in final edited form as: J Mol Cell Cardiol. 2013 Oct 23;66:27–40. doi: 10.1016/j.yjmcc.2013.10.010

A) Diagram of AKAP-Lbc-ΔPKD (top) and AKAP-Lbc (WT, bottom). AKAP-Lbc-ΔPKD lacks the C-terminus of AKAP-Lbc, where the PKD1 binding region resides, due to fusion of a β-Geo cassette. B) Quantification of AKAP-Lbc-WT and AKAP-Lbc-ΔPKD atrial and ventricular expression by RT-PCR using AKAP-Lbc-specific primers 6485 forward and 6560 reverse. C) Control RT-PCR quantifying AKAP-Lbc-ΔPKD atrial and ventricular expression using βGeo-specific primers. D) AKAP-Lbc-PKD binding is abolished and associated activity is significantly reduced in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous AKAP-Lbc was immunoprecipitated from heart lysate and associated PKD activity was measured by in vitro kinase assay. Results presented show mean kinase activity per IP ± SEM after control IgG IP background activity has been subtracted. Western blot shows corresponding levels of AKAPLbc and PKD in samples and lysates used for PKD activity measurement. E) AKAP-Lbc-PKA binding and associated PKA activity is similar in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous AKAP-Lbc was immunoprecipitated from heart lysate and associated PKA activity was measured by in vitro kinase assay. Western blot shows corresponding levels of AKAP-Lbc and PKA (PKAc; catalytic subunit) in samples and lysates used for PKA activity measurement. F) Total cardiac PKD activity is significantly reduced in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous PKD was immunoprecipitated from heart lysate and kinase activity was measured in vitro. Results presented show mean kinase activity per IP ± SEM after control IgG IP background activity has been subtracted. Western blot shows corresponding levels of PKD1 in samples and lysates used for PKD activity measurement. G) Total cardiac PKA activity is similar in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous PKAc was immunoprecipitated from heart lysate and kinase activity was measured in vitro. Results presented show mean kinase activity per IP ± SEM after control IgG IP background activity has been subtracted. Western blot shows corresponding levels of PKAc in samples and lysates used for PKA activity measurement. H) AKAP-Lbc Rho-GEF activity is similar in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous AKAP-Lbc was immunoprecipitated from heart lysate and RhoA-GEF activity was measured by in vitro assay. Western blot shows corresponding levels of AKAP-Lbc in samples used for activity measurement. Differences in quantitative variables were examined by Student's t-test. ** = p < 0.01. N.S = no significant difference between groups. All assays were performed in triplicate for 3 independent experiments.

A) Diagram of AKAP-Lbc-ΔPKD (top) and AKAP-Lbc (WT, bottom). AKAP-Lbc-ΔPKD lacks the C-terminus of AKAP-Lbc, where the PKD1 binding region resides, due to fusion of a β-Geo cassette. B) Quantification of AKAP-Lbc-WT and AKAP-Lbc-ΔPKD atrial and ventricular expression by RT-PCR using AKAP-Lbc-specific primers 6485 forward and 6560 reverse. C) Control RT-PCR quantifying AKAP-Lbc-ΔPKD atrial and ventricular expression using βGeo-specific primers. D) AKAP-Lbc-PKD binding is abolished and associated activity is significantly reduced in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous AKAP-Lbc was immunoprecipitated from heart lysate and associated PKD activity was measured by in vitro kinase assay. Results presented show mean kinase activity per IP ± SEM after control IgG IP background activity has been subtracted. Western blot shows corresponding levels of AKAPLbc and PKD in samples and lysates used for PKD activity measurement. E) AKAP-Lbc-PKA binding and associated PKA activity is similar in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous AKAP-Lbc was immunoprecipitated from heart lysate and associated PKA activity was measured by in vitro kinase assay. Western blot shows corresponding levels of AKAP-Lbc and PKA (PKAc; catalytic subunit) in samples and lysates used for PKA activity measurement. F) Total cardiac PKD activity is significantly reduced in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous PKD was immunoprecipitated from heart lysate and kinase activity was measured in vitro. Results presented show mean kinase activity per IP ± SEM after control IgG IP background activity has been subtracted. Western blot shows corresponding levels of PKD1 in samples and lysates used for PKD activity measurement. G) Total cardiac PKA activity is similar in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous PKAc was immunoprecipitated from heart lysate and kinase activity was measured in vitro. Results presented show mean kinase activity per IP ± SEM after control IgG IP background activity has been subtracted. Western blot shows corresponding levels of PKAc in samples and lysates used for PKA activity measurement. H) AKAP-Lbc Rho-GEF activity is similar in AKAP-Lbc-ΔPKD mice, compared to WT mice. Endogenous AKAP-Lbc was immunoprecipitated from heart lysate and RhoA-GEF activity was measured by in vitro assay. Western blot shows corresponding levels of AKAP-Lbc in samples used for activity measurement. Differences in quantitative variables were examined by Student's t-test. ** = p < 0.01. N.S = no significant difference between groups. All assays were performed in triplicate for 3 independent experiments.