3xflag myc cmv 26 vector

Stably transfected Human Embryonic Kidney (HEK293T) cells, expressing 3xFlag-myc-CMV-26 vector (Sigma-Aldrich, UK) containing full-length human EPAC1, as described [39 (link),40 (link)], were grown in Dulbecco's modified Eagle's medium (DMEM), 10% (v/v) foetal bovine serum (Sigma-Aldrich, UK), 1% (v/v) of GlutaMAX supplement (Sigma-Aldrich, UK) and 1% (v/v) penicillin/streptomycin (Sigma-Aldrich, UK) and incubated at 37 °C in 5% (v/v) CO₂. Selection of stable cell lines was maintained by addition of 1 mg/ml G418 (Sigma-Aldrich, UK) to the growth medium. The human monocytic cell line THP-1 was cultured in RPMI 1640 medium (Fisher Scientific, UK) containing 10% (v/v) foetal bovine serum (Sigma-Aldrich, UK), 1% (v/v) penicillin/streptomycin (Sigma-Aldrich, UK) and 2 mM glutamine at 37 °C in 5% (v/v) CO₂. HUVECs were grown in EGM2 (PromoCell) at 37 °C and 5% (v/v) CO₂. Cells were passaged weekly to a maximum of six passages.

Stably transfected Human Embryonic Kidney (HEK293T) cells, expressing either 3xFlag-myc-CMV-26 vector (Sigma-Aldrich, UK) containing full-length human EPAC1 or vector alone, were prepared by Dundee Cell Products (Dundee, UK). HEK293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) 10% (v/v) foetal bovine serum (Sigma-Aldrich, UK), 2% (v/v) glutamine (Sigma-Aldrich, UK) and 2% (v/v) penicillin/streptomycin (Sigma-Aldrich, UK) and incubated at 37 °C in 5% (v/v) CO₂. Selection of stable cell lines was maintained by addition of 400 mg/ml G418 (Sigma-Aldrich, UK) to growth medium.

Human Embryonic Kidney (HEK293T) cell lines stably expressing either 3xFLAG-myc-CMV-26 vector (Sigma-Aldrich, UK) containing full-length human EPAC1, or vector alone, were prepared by Dundee Cell Products (Dundee, UK). COS1 and HEK293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) 10% (v/v) foetal bovine serum (Sigma-Aldrich, UK), 2% (v/v) glutamine (Sigma-Aldrich, UK) and 2% (v/v) penicillin/streptomycin (Sigma-Aldrich, UK) and incubated at 37 °C in 5% (v/v) CO₂. Selection of stable cell lines was maintained through addition of 400 μg/ml G418 (Sigma-Aldrich, UK) to the cell culture medium. Human umbilical cord endothelial cells (HUVECs) were grown in Endothelial Growth Medium MV2, supplemented with the MV2 supplement pack (Promocell, Heidelberg, Germany) and 1% (v/v) penicillin/streptomycin. Experiments using HUVECs were performed between Passages 1 and 6.