Increased amount of ATF2, c-Jun, and ATF3 proteins and their association in HUVECs stimulated by homocysteine.

(A) Cultured HUVECs were exposed to 3 mmol/L homocysteine for the indicated time, and their whole-cell extracts (20 μg) were subjected to Western blot with anti-ATF2 or anti–c-Jun antibody as described in “Materials and methods.” (B) Nuclear extracts prepared from the control and homocysteine-stimulated cells for 4 hours were immunoprecipitated with anti-ATF2 antibody as described in “Materials and methods.” The resultant complexes were separated on a 12% SDS-PAGE under nonreducing conditions, under which IgG migrated as a protein of large molecular mass, and analyzed by Western blotting with anti-ATF2 (lanes 1-3), anti–c-Jun (lanes 4-6), or anti-ATF3 (lanes 7-9) antibody as probe. Lanes 1, 4, and 7 were results of the homocysteine-treated nuclear extracts immunoprecipitated with nonimmune IgG.