Figure 4

P-Rex1 plays an essential role in synergism of Gβγ- and PI3K-dependent migration. LPA- and EGF-induced dose-dependent activation of Rac for 5 min was determined by measuring the GTP form of Rac as described in 'Materials and methods' (A, B). (C) A549 cells were pretreated for 20 min with various inhibitors such as Ki16425 (2 nM), gallein (10 µM), AG1478 (100 nM), LY294002 (10 µM) or gallein together with AG1478 or LY294002, followed by stimulation with LPA (10 nM) and EGF (50 pg/ml) for 5 min. (D) After knock-down of P-Rex1, activation of Rac was determined by measuring the GTP form of Rac and expression of P-Rex1 was determined by RT-PCR. (E) Motility after silencing P-Rex1 was determined by migration assay for 10 h. (F) LPA (10 nM)- and EGF (50 pg/ml)-induced cancer cell invasion for 24 h was measured as described in 'Materials and methods' section. ^*P < 0.05. (G) Cells were stimulated with LPA (10 nM), EGF (50 pg/ml) or together with LPA and EGF for 24 h. MMP-2 protein expression and gelatinolytic activity were analyzed by western blotting (WB) and gelatin zymography (Zym), respectively.