PKA and AKAP220 form a complex with junctional-associated proteins VE-cadherin and ß-catenin.

<p>A complex consisting of PKA, ß-catenin, VE-cadherin and AKAP220 was detected in MyEnd either by pulling down VE-cadherin (A) or PKA (B). Immunoprecipitated proteins and their binding partners were detected in the lysates used for immunoprecipitation. The lysates were loaded at the first lane and denoted as an “Input”. Immunoprecipitation without antibody (IP: Beads, no Ab) was used as a negative control. Every other lane represents proteins in complex under different experimental conditions. (C) To test the role of PKA compartmentalization on the stability of the determined complex, PKA pull down was performed in cells treated either with inhibitory or with the respective scrambled peptide. Additionally, the effect of F/R on complex stability was analyzed. (D) summarizes data collected from three independent immunoprecipitation experiments. Firstly, intensity readings for each band were background corrected. Secondly, data was modified by substraction of “non-specific background intensities” detected in IP without Ab (negative control). Since PKA was used to pull-down the complex, signal intensity of the immunoprecipitated proteins was normalized to the band intensity of PKA (represented as fold changes of PKA staining intensity). As a final, the data was presented as percent of IP: PKA, TAT-Ahx-mhK77. The analysis revealed decreased complex association of ß-catenin, VE cadherin and AKAP220 after TAT-Ahx-AKAPis treatment. In contrast, F/R application led to stabilization of the complex indicated by more prominent intensities for bands representing ß-catenin, VE-cadherin and AKAP220. Noncontiguous bands run on the same gel are separated by a black line. Images are representative of three independent experiments (N = 3).