Figure_2.tif (1.35 MB)

The gene-trap induced truncations of AKAP13 disrupt the expected protein domains.

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posted on 26.04.2013, 00:39 by Matthew J. Spindler, Brian T. Burmeister, Yu Huang, Edward C. Hsiao, Nathan Salomonis, Mark J. Scott, Deepak Srivastava, Graeme K. Carnegie, Bruce R. Conklin

(A) Expression constructs corresponding to the AKAP13 gene-trap events were generated using V5-tagged AKAP-Lbc truncation mutants. (B-D) These expression constructs were transfected into HEK293 cells and protein complexes were co-immunoprecipitated using anti-V5 antibody. (B) Rho-GEF activity was measured after immunoprecipitation (IP). Both AKAP-Lbc-ΔGEF and -ΔBrx had disrupted Rho-GEF activity, compared to AKAP-Lbc-WT and -ΔPKD. Immunoblotting (IB) for AKAP-Lbc-V5 with anti-V5 antibody confirmed that the AKAP-Lbc truncation mutants were expressed and immunoprecipitated at an equivalent extent. (C) Protein kinase D (PKD) activity was measured following IP. The AKAP-Lbc-ΔPKD, -ΔGEF, and -ΔBrx protein complexes lacked PKD activity compared to AKAP-Lbc-WT. Immunoblotting for GFP-PKD1 with anti-GFP antibody confirmed that only AKAP-Lbc-WT bound PKD1. The bottom gel image confirmed that GFP-PKD1 was expressed at the same level in all conditions. (D) Protein kinase A (PKA) activity was measured after IP. All AKAP-Lbc truncation mutants immunoprecipitated PKA activity and bound PKAc. The means and standard deviations are graphed for three independent experiments. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). *, p<0.05; ***, p<0.001.