BAECs were pretreated with vehicle (Me2SO (DMSO), or 1 μmol/liter herbimycin (A), or 10 μmol/liter PP2 (B) for 30 min before exposure to flow for 15 min. Phosphorylation of Gab1, Akt, and eNOS in cell lysates were detected by immunoprecipitation (IP) and immunoblotting (IB) with phosphospecific antibodies. The same blots were stripped and reprobed with antibody detecting total Gab1, Akt, and eNOS to monitor equal loading of samples. Results are representative for three independent experiments.