Odissey clx scanner

Two hundred thousand rBCEC4 grown in 6 well plates were incubated with control medium or EPC-CM for 15 min. The phosphorylation inhibitors [5 µM] LY294002 or [10 µM] PD98059 were administered 30 min prior and during exposure to EPC-CM. Following treatments, the cells were washed 3 times with cold HBSS and lysed in RIPA buffer (Pierce Biotechnology, Rockford, IL, USA) containing Protease Inhibitor Cocktail Set V (Calbiochem),1 µM Sodium Orthovanadate (Sigma), 10 µM PMSF (Sigma) and 1 µM Leupeptin (Sigma). After centrifugation the supernatant was collected and the protein concentration was estimated using the BCA kit (Pierce Biotechnology). Equal amount of proteins were resolved on a 12% polyacrylamide gel followed by overnight transfer and hybridization with primary antibodies: mouse anti total AKT (Cell Signaling), rabbit anti pAKT (Cell Signaling), mouse anti total ERK (Millipore), rabbit anti pERK (Millipore). As secondary antibodies Dylight-800 anti mouse and Dylight-680 anti rabbit were used. Immunoreactive bands were visualized using an Odissey CLx scanner (LI-COR) equipped with LI-COR Image. Quantification was performed on grey values converted images using the software ImageJ.