SU1498

SU1498 akzentuiert die Akkumulation von phosphoryliertem ERK in Endothelzellen und hemmt dessen Kinaseaktivität, während es keine Auswirkung auf die Phosphorylierung von Akt hat. Diese Verbindung beeinflusst die Phospho-ERK-Translokation zum Zellkern nicht[1].

• Zelllinien

  HUVECs

• Konzentrationen

  10 μM

• Inkubationszeit

  15 min

• Methode

  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.