Brk mediates cell type-specific Met and Ron receptor signaling to cell migration. (a) In vitro kinase assays were performed in HaCaT cells as previously described. Cells were serum-starved for 24 hr then treated with either vehicle (water), MSP (80 ng/ml), HGF (50 ng/ml), or heregulin (25 ng/ml) for 15 min. Purified recombinant Sam68 was used as a substrate to measure Brk kinase activity. IgG control images were derived from the same experiment, Western blot, and film exposure time as the experimental lanes shown. Boyden chamber migration assays were performed in HaCaT (b), MDA-MB-231 (c), and T47D (d) cells expressing non-targeting (control) siRNA or Brk siRNA. Cells were treated with either vehicle (water) or 80 ng/ml MSP as the chemoattractant (in the lower chamber). 50 ng/ml HGF was used as a positive control chemoattractant for 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 or MSP treated cells expressing control siRNA. Double asterisks (**) denote significance between control and Brk siRNA with either HGF or MSP used as chemoattractants. Results were confirmed in three independent experiments. Cells treated at various time points with 80 ng/ml MSP were subjected to Western blotting with total ERK5 specific antibody. HGF treatment was used as a positive control for ERK5 activation (insets).