Figure 5.

PKA inhibits endothelial cell migration by suppressing Src activity. (A) Anti-Src pY529 and anti-total Src immunoblots of lysates from endothelial cells treated with or without forskolin (+forskolin). pY529Src/total Src ratio was determined by densitometry, *P < .05. (B) Graph: ³²P incorporation into a Src substrate (KVEKIGEGTYGVVYK) by Src immunoprecipitated from cells treated with and without forskolin, expressed as pmol³²P incorporated per minute ± SEM, *P < .05. Blot: Loading control showing Src immunoprecipitates blotted with anti-Src antibodies. (C) Mean number of endothelial cells migrating ± SEM after transduction with control virus (CA10) or virus expressing truncated Src, Src251 (CA10Src251), or after treatment with culture medium or medium containing 10μM PP2 (Src inhibitor) or 10μM PP3 (negative control), *P < .05. Blot: Src251 expression demonstrated by immunoblotting of cell lysates with anti-Src antibodies. (D) Mean number ± SEM of migrated control transfected endothelial cells (dark bars) or SrcY529A transfected endothelial cells (SrcA, white bars) treated with or without forskolin or transfected with PKACat or GFP. Blot: Transfected cell lysates immunoblotted with anti-Src and anti-GFP antibodies. (E) Mean number of migrating src-fyn-yes^−/− fibroblasts (syf) and syf fibroblasts stably expressing Src (syf/Src) that were treated with medium (−forskolin) or 20 μg/mL forskolin (+forskolin). *P < .05.