TI - Kinetics of ERK1/2 activation by 17beta-estradiol in MCF-7 cells enriched and MCF-7 cells depleted for membrane ER-alpha . AB - During the optimization of the fixed cell -based enzyme -linked immunosorbent assay for ERK activation , we established the optimal cell density for a 10 min EGF treatment , which is known to result in substantial PHOSphorylation of ERK1/2 . Both the controls and EGF-treated cells exhibited increased pERK1/2 with increased cell number ( Fig 6a , main graph ) . As we expected , normalizing pERK1/2 values to the number of cells ( CV absorbance at 590 nm ) did not significantly change the ratio values except in the case of the highest number of cells plated , verifying that cells plated in the density range of CV values 0.2-0.6 could be used . Partly because others have shown that ERKs can be activated by mechanical stimulus in MCF-7 cells [29] , we tested ethanol-treated controls over the same time course ( 3-60 min ) . A pronounced decrease in ERK1/2 PHOSphorylation was seen with time ( Fig 6b ) , and so appropriate controls were performed for each time point in all subsequent experiments . In mERhigh MCF-7 cells , ERK1/2 activation with 1 pmol/l E2 was fast and transient ( Fig 7a ) . The maximal activation was achieved after 10 min , followed by a rapid decline in PHOSphorylated ERK1/2 . However , continued incubation with E2 ( 1 hour ) resulted in the reactivation of ERK1/2 . To test whether this signal was initiated at the membrane , the impeded ligand E2-peroxidase was applied to the cells at steroid concentrations that approximated the levels applied as free steroid in the previous experiments ( Fig 7b ) . To eliminate any free steroid present , just before use we pretreated the E2-peroxidase with dextran-coated charcoal under conditions that remove more than 99% of free hormone [9] . The resulting maximal level of ERK1/2 activation was slightly higher than for treatment with the free ligand , but the peak time of activation was the same . Again , a recurrent later ERK activation was observed . Cells with lower levels of mER-alpha ( Fig 7c ) also had the capacity for fast and transient activation of ERK1/2 , but this smaller activation peak appeared at 6 min after 1 pmol/l E2 treatment . The levels of phosphorylaTED ERK declined between 10 and 30 min of E2 treatment as they had with mERhigh cells ; however , at longer incubations ( 1 hour ) no reactivation was seen but rather a further ERK1/2 apparent dePHOSphorylation was observed . This implies that higher levels of mER-alpha associated with more robust early ERK activation are also responsible for the sustained ERK reactivation at the later stage . The inhibitor of the upstream MEK1/2 , namely U0126 , was effective in inhibiting ERK1/2 activation in both types of MCF-7 cells ( Fig 6a and 6c , insets ) , verifying that the values we measured in our plate assay were from MEK -PHOSphorylated ERK . In the MDA-MB-231 ER-alpha-negative cell line ( Fig 7d ) , E2 could not significantly activate ERK1/2 , confirming that ER-alpha is necessary for ERK activation during this 60 min time period .