SU1498

SU1498 accentuates accumulation of phosphorylated ERK in endothelial cells and inhibits its kinase activity while having no effect on phosphorylation of Akt. SU1498 does not affect phospho-ERK translocation to nucleus^[1].

Cells are inoculated into the wells of 6-well culture plates (3 × 10⁵ cells/well) and grown for 16 h in DMEM-based medium at 37 °C in the atmosphere of 5% CO2. After an overnight starvation (0.5% fetal bovine serum), the medium is removed, and the cells are washed with PBS and overlaid with 0.4 ml of DMEM. From this point on, all incubations are done at 25 °C. The inhibitors (SU1498, 10 μM; AG1433, 10 μM; AG1296, 25 μM; PD153035, 1 μM) are added and followed 15 min later by the growth factors: SPP, 1 μm; VEGF, 100 ng/ml; HGF, 100 ng/ml; bFGF, 10 ng/ml; EGF, 10 ng/ml. Forskolin (10 μm), a gratuitous inducer of adenylyl cyclase, is used as a control because of its known ability to enhance ERK phosphorylation. After a 10-min incubation, the medium is siphoned off, and the cells are lysed with 100 μl of lysis buffer. Samples (10 μl) of each lysate are fractionated by SDS-PAGE, and proteins are transferred to nitrocellulose and probed with anti-phospho-Akt or anti-phospho-ERK1/2 antibody. To show comparable loading, anti-actin antibody is used.