Brk kinase activity is not required for HGF-induced cell migration. (a) HaCaT and MDA-MB-231 (231) cells were transiently cotransfected with non-targeting (control) siRNA or Brk siRNA targeting the 3′-UTR of Brk mRNA and flag-tagged pCMV (vector), wt-Brk, or km-Brk. Western blot analysis was performed using total ERK5 and Brk-specific antibodies to identify endogenous Brk and flag-tagged Brk. p38 MAPK protein was used as a loading control. Boyden chamber migration assays were performed in HaCaT (b) and MDA-MB-231 (c) cells transiently coexpressing nontargeting siRNA or Brk 3′-UTR siRNA and either flag-tagged pCMV, wt-Brk, or km-Brk. Vehicle (water) or 50 ng/ml HGF were used as chemoattractants in the lower chamber. 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 Brk 3′-UTR siRNA cells coexpressing wt- or km-Brk. Results were confirmed in three independent experiments.