Mevalonate rescues 15(S)-HETE–induced farnesylation, membrane translocation and activation of Rac1 in HDMVECs and the migration and tube formation of these cells from inhibition by simvastatin. (A-B) Quiescent HDMVECs were treated with and without 0.1μM 15(S)-HETE for the indicated time periods or 60 minutes in the presence and absence of 10μM simvastatin in combination with or without 50μM mevalonate and either whole cellular extracts or the cytoplasmic and membrane fractions were prepared. Rac1 farnesylation was measured by immunoprecipitation of an equal amount of protein with anti-farnesyl antibodies followed by immunoblotting with anti-Rac1 antibodies. Rac1 activation was measured by pull-down assay. Rac1 and Na/K ATPase levels were measured by Western blotting using their specific antibodies. (C) All the conditions were the same as in panel B, except that cells were fixed, permeabilized and immunostained for Rac1 using anti-Rac1 antibodies followed by probing with Alexa Fluor 568–conjugated secondary antibodies. (D-E) HDMVECs were treated with or without 10μM simvastatin in the presence and absence of 50μM mevalonate, trypsinized, rinsed with TNS and subjected to 15(S)-HETE (0.1μM)–induced tube formation (D) or migration (E). The bar graphs in panels A, D and E represent the mean ± SD values of 3 independent experiments. *P < .01 versus control; **P < .01 versus 15(S)-HETE; ***P < .01 versus simvastatin + 15(S)-HETE. C indicates cytoplasmic fraction; M, membrane fraction; ss, simvastatin; and ML, mevalonate.