HGF-induced cell migration requires ERK5 in breast cancer cells. Boyden chamber migration assays were performed on HaCaT (a) and MDA-MB-231 (b) cells as previously described. Cells were transiently transfected with either non-targeting (control) siRNA or ERK5 siRNA. Lower chambers were supplemented with either vehicle (water) or 50 ng/ml HGF as the chemoattractant. Boyden chamber migration assays were performed for 6 hr. Error bars indicate the mean (plus standard deviation) of triplicate measures of cell migration. Single asterisks (*) denote significance (P < 0.05) determined by an unpaired Student's t-test between vehicle and HGF treated cells expressing control siRNA. Double asterisks (**) denote significance between control and ERK5 siRNA with HGF used as the chemoattractant. Results were confirmed in three independent experiments. Cells treated for 15 min with HGF were subjected to Western blotting; ERK5 activation and protein expression were determined using total ERK5 specific antibodies (insets).