Brk expression enhances MDA-MB-435 cell migration. (a) MDA-MB-435 cells were transiently transfected with pRcCMV (vector), wt-, or km-Brk. Boyden chamber migration assays were performed with cells using either vehicle (water) or 50 ng/ml HGF as the chemoattractant. A concentration of 20% FBS was included as a positive control for robust migration. 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. Double asterisks (**) denote significance between cells expressing vector and wt-Brk with HGF as the chemoattractant. 20% FBS was used as a positive control chemoattractant. (b) MDA-MB-435 cells were transiently transfected with PRcCMV (vector), wt-, or km-Brk. Cells were treated for 15 min with vehicle (water) or 50 ng/ml HGF. Whole cell lysates were subjected to Western blotting with total-ERK5, Brk, and total-p38 antibodies. Brk-specific antibodies were used to demonstrate equal transfection efficiency and total endogenous p38 MAPK served as a loading control.