Akt mediates EPC adhesion. (A) M07e cells stimulated with the KitL were lysed. (B) EPCs let to adhere to fibroblasts were lysed. Cell extracts from M07e cells (A) and from EPCs adherent to fibroblasts (B) were analyzed with anti–p–c-Kit, anti–c-Kit, anti–p-ERK1/ERK2 MAPK, anti-ERK1/ERK2, anti–p-Akt, or anti-Akt antibodies as indicated. (C) EPCs transfected with an Akt siRNA or with the scrambled sequence (scramble) were lysed. The filter was IB with an anti-Akt and anti–β-actin antibody. (D) Adhesion on fibroblasts was performed using EPCs depleted or not of the endogenous Akt. Experiments were also performed by preincubating EPCs with the Akt kinase inhibitor. Adherent cells were counted for statistical analysis (data are the mean ± SD; *P < .05 control vs experimental groups). (E) EPCs were transfected with the c-Kit mutants or the empty vector. After 24, 48, and 72 hours of transfection, EPCs were let to adhere to fibroblasts for 30 minutes and lysed. The filters were IB with an anti–p-Akt and an anti-Akt antibody. M07e cells were used as positive control (+). (F) Fibroblasts and activated CDC-HMEC-1 cells were used for adhesion of EPCs transfected (72 hours) with the Y821F and Y719F mutants or the empty vector. Adherent cells were counted for statistical analysis (data are the mean ± SD; *P < .05 control vs experimental groups). Five different experiments were performed with similar results.