Figure 4.

The MEK/ERK pathway is involved in the aggregation of WT cells. (A) Cells(5 × 10⁵) were seeded in 60 mm plates. After IPTG induction for 24 h, the signaling pathway inhibitors were added and incubated for another 24 h. The p-ERK, ERK, p-AKT, AKT, Ras, Aurora-A and β-actin were detected by western blotting. The level of each protein was quantified by a densitometer. The expression level of each protein in Vector cells without IPTG induction was set as 1.0 fold. (B) Cells(5 × 10⁵) were seeded in 60 mm plates.After transfection of WT cells for 6 h with pHA-Vector (5 μg), pHA-RalA183A (5 μg) or pHA-RalA194A (5 μg), IPTG was added and incubated for another 48 h. The total cell lysates (500 μg) were used to detect the activity of Ral A by Ral A pull-down assay. (C) Cells (5 × 10⁵) were seeded in 60 mm plates. After IPTG induction for 24 h, the inhibitors were added and incubated for another 24 h. For Aurora-A RNAi and RalAS194A, after transfection of cells for 6 h with Aurora-A siRNA (5 μg) or pHA-RalA194A (5 μg), IPTG was added and incubated for another 48 h. a.WT cells treated with IPTG. b.WT cells treated with IPTG/Aurora-A siRNA c.WT cells treated with IPTG/FTI-277 d.WT cells treated with IPTG/PD98059 e.WT cells treated with IPTG/LY294002 f.WT cells treated with IPTG/pHA-RalA194A. FTI-277, a farnesylation inhibitor of Ras; PD98059, an inhibitor of MEK kinase; LY294002, an inhibitor of PI3K kinase.