Research Paper Volume 14, Issue 17 pp 7093—7108

Figure 4. Effect of MSP on RON phosphorylation and activation of downstream signal molecules. 5637 cells (2 × 10⁶ cells per 60-mm culture dish) were incubated in RPMI-1640 containing 1% FBS overnight and then stimulated for 30 mins with MSP (5nM). Western blot analysis measured the effects of MSP on the phosphorylation levels of RON, Erk1/2, RSK2 and CREB (A). 5637 cells (2 × 10⁶ cells per dish) in RPMI-1640 with 1% of FBS were first treated with Small chemical inhibitors specific to RON (CP-1, 300 nM) (B), Erk1/2 (PD98059, 150 μM) (C), and RSK (SL0101, 50μM) (D) for 12 h, respectively, followed by stimulation with MSP (5nM). Cells were collected 30 mins after stimulation. Phosphorylated RON, Erk1/2, RSK2 and CREB were directly detected by Western blot analysis, respectively. Non-phosphorylated proteins were also determined as the loading controls. (E–H), Quantitative analysis, *P<0.05, **P<0.01 and ***P<0.001 VS. normalized to their own total protein group. Data are shown as mean ± SEM, from one of 3 independent experiments.