FIGURE 5.

ERK5 mediates breast cancer cell migration through ERK5-Sam68 complex dissociation. MDA-MB-231 cells were pretreated with either 10 μm U0126 for 30 min (A) or 10 nm or 10 μm PD (B) and then treated with either vehicle or 50 ng/ml HGF for 15 min. ERK1/2 and ERK5 activation was evaluated using phospho-specific and total specific antibodies, respectively; total ERK1/2 served as a loading control. C, MDA-MB-231 cells were assayed for migration in response to HGF in the presence or absence of either low (10 nm) or high (10 μm) concentrations of PD. D, Boyden migration assays were performed using cells transiently expressing control or ERK5 siRNA (inset shows ERK5 and a nonspecific protein loading control) and stimulated with HGF. * denotes significance (p < 0.05) relative to vehicle and between indicated groups, as determined by unpaired Student's t test. § denotes no statistical difference relative to vehicle. E, cells were treated with either vehicle or 50 ng/ml HGF for 15–30 min. ERK5 was immunoprecipitated (IP) from whole cell lysates using total ERK5-specific antibodies. ERK5 immunoprecipitates were then subjected to Western blotting (IB) with total ERK5 and Sam68-specific antibodies. F, cells were pretreated for 30 min with low (10 nm) or high (10 μm) concentration of PD, followed by 15 min of HGF treatment. ERK5 was immunoprecipitated from whole cell lysates using total ERK5-specific antibodies and Western-blotted with ERK5 and Sam68-specific antibodies. ERK1/2 and ERK5 activation was evaluated using phospho-specific and total specific antibodies, respectively.