Western blot analysis

The total and phosphorylated ERK, p38 MAPK, JNK, PI3K, Akt, and mTOR protein in HUVECs was detected by Western blot analysis, besides the protein levels of bcl-2 and Bax. As an internal control, GAPDH was detected. The primary antibodies for these proteins were all acquired from Cell Signaling Technology. Densitometric analysis of the Western blots was performed using Image Lab™ Software (Bio-Rad Version 3.0; Bio-Rad Laboratories Inc., Hercules, CA, USA). The relative values of the samples were measured by normalizing all data to the respective control samples of each experiment.