Intact Met signaling in T47D cells. (a) Real-time quantitative RT-PCR was performed to determine the mRNA expression levels of Met and Ron receptors in T47D, MDA-MB-231, and HaCaT cells. Error bars indicate the mean (plus standard deviation) of Met and Ron mRNA, normalized to β-actin, measured in triplicate cultures. Results were confirmed in three independent experiments. Met protein levels in all three cell lines as measured by Western blotting (insets) (b) T47D, HaCaT and MDA-MB-231 cells were serum-starved for 24 hr and then treated with 50 ng/ml HGF or 80 ng/ml MSP for 5 to 90 min. Western blot analyses were performed on whole cell lysates using ERK5-specific phospho- and total antibodies. ERK1/2 protein expression was used as a loading control. (c) T47D cells were serum-starved for 24 hr then treated with 20 or 50 ng/ml HGF for 10 min. Whole cell lysates were subjected to Brk immunoprecipitation and in vitro kinase assay using purified recombinant Sam68 as described in Material and Methods.