Tβ4 induces phosphorylation of JNK1, ERK, and phospho-c-Jun protein. Confluent EA.hy 926 cells were treated with increasing concentrations of Tβ4 (0-80 nM) for 20 minutes or overnight in the case of ERKs and JNK1 or c-Jun, respectively. Total protein was harvested in 2 × sodium dodecyl sulfate (SDS) buffer. After polyacrylamide gel electrophoresis (PAGE) separation and transfer to polyvinylidene difluoride membranes, Western blot analysis was performed with an antibody to ERK, JNK, and phospho-(p)-c-Jun (Ser63), and enhanced chemiluminescence was performed for detection of bound secondary antibody. All data are representative of 3 replicate experiments.