HUVEC MAPK activation by free and membrane-impermeant forms of estrogen. Serum-deprived HUVEC monolayers were E2 (50 ng/ml)-treated for the indicated times (A) or treated for 15 min with either E2 (50 ng/ml), E2coBSA (395 ng/ml, equivalent to 50 ng/ml of E2), or E2hsBSA (308 ng/ml, equivalent to 50 ng/ml of E2) (B). Subsequently, activated ERK1/2, immunoprecipitated from cell lysates, was used in an in vitro kinase assay with ELK-1 peptide substrate. Kinase reaction samples were immunoblotted with a phospho-specific anti-ELK-1 antibody. Controls were treated with 5% BSA containing media alone (C) or the addition of purified activated MAPK to the kinase reaction (C+).