ERK5 siRNA expressing cells signal to ERK1/2 to mediate HGF-induced cell migration in HaCaT cells. (a) HaCaT cells expressing non-targeting (control) siRNA or ERK5 siRNA were pre-treated for 30 min with 5 μM U0126, then treated for 15 min with HGF and Western blot analysis was performed using phospho- and total-specific antibodies specific for ERK1/2 or total ERK5. (b) Boyden chamber migration assays were performed using HaCaT cells expressing either non-targeting (control) siRNA or ERK5 siRNA. Cells were treated with either vehicle (water) or 50 ng/ml HGF and in the presence or absence of 5 μM U0126 in the lower and upper chambers. 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 conditions in either control or ERK5 siRNA expressing cells. Results were confirmed in three independent experiments. (c) MDA-MB-231 cells were transiently transfected with control siRNA or ERK5 siRNA. Cells were serum starved for 24 hr and then treated with vehicle or HGF at 15 min. Whole cell lysates were subjected to Western blotting with total-ERK5, phospho-ERK1/2 or total-ERK1/2 antibodies. ERK1/2 protein expression served as a loading control.