HGF-induced cell migration requires Brk/ERK5 complexes in breast cancer cells. (a) HaCaT and MDA-MB-231/(231) cells were treated with either vehicle or HGF for 15-30 min (30 min for 231 cells). ERK5 was immunoprecipitated from whole cell lysates using total ERK5-specific antibodies. ERK5 immunoprecipitates were then subjected to Western blotting with Brk-specific antibodies. Lysates (10% input) were Western blotted with total ERK5 and Brk-specific antibodies. (b) COS cells were transiently transfected with flag-tagged pCMV (vector), wt or kinase dead (km) Brk. ERK5 was immunoprecipitated from whole cell lysates using ERK5-specific antibodies and Brk was identified using flag-specific antibodies. Lysates (10% input) were Western blotted with flag antibody to indicate the efficiency of ERK5/Brk co-association.