Human bone osteosarcoma epithelial cells (U2OS) stably transfected to specific FLAG-tagged EPAC1 have been a present from Professor Holger Rehmann (Flensburg College of Utilized Sciences, Germany). HUVECs have been used for experiments at no older than passage 6. Cells have been cultured within the development medium, comprising Endothelial Cell Progress Basal Medium supplemented with Endothelial Cell Progress Medium SingleQuots Dietary supplements (excluding antibiotics), in 75 cm 2, tissue tradition handled flasks, at 37 ☌, in a humidified ambiance containing 5% (v/v) CO 2. Cell traditionĬryopreserved major human umbilical vein endothelial cells (HUVECs) in addition to devoted medium and development dietary supplements, have been purchased from (PromoCell, Heidelberg, Germany).

Compounds SY006, SY007 and SY009 have been synthesised as beforehand described 19. Chemical synthesisĬompounds I942, PW0381, PW0521 and PWO577 have been synthesised as beforehand described (compounds 3, 25g, 25q, 25n, respectively, in 21). Rap1A/Rap1B (26B4) rabbit mAb, EPAC1 (DE3) mouse mAb and STAT3/phospho-STAT3 (Tyr 705) antibodies have been bought from Cell Signaling Expertise, Danvers, MA, USA. Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) sodium salt, 0.33% impartial purple answer, dimethyl sulfoxide (DMSO) for molecular biology and anti-Rabbit IgG, HRP conjugate, have been obtained from Sigma-Aldrich, St.

5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), Alamar blue cell viability reagent and SYPRO™ Orange protein stain have been bought from ThermoFisher Scientific, Waltham, MA, USA. 8-(4-chlorophenylthio)-2′- O-methyladenosine-3′,5′-cyclic monophosphate (D-007) was bought from Biolog Life Science Institute, Bremen, Germany.

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