Fig. 4. Activation of MCF-7 cell ERK by MDA-MB-231 cell CM. MDA-MB-231 cells were allowed to condition serum-free medium for 24 hours. The CM was subjected to centrifugation at 15,000 g for 10 minutes to remove debris and then added to cultures of MCF-7 cells that had been serum-starved for 12 hours. Some samples of CM were pre-incubated with uPA-specific antibody (25 µg/ml) or mouse nonimmune IgG (25 µg/ml) for 20 minutes at 37°C before being added to the MCF-7 cell cultures. ERK phosphorylation was assessed 1 minute after addition of the CM.