EMSAs of complexes formed with the ATF/CRE motif of the ATF3 gene promoter.

(A) Nuclear extracts (NEx) from the control or homocysteine-treated HUVECs were subjected to EMSAs as described in “Materials and methods.” Lane 1, probe only; lane 2, control extract; lanes 3, 4, and 5, homocysteine-treated extract incubated in the absence (3) or presence of 25- (4) or 50-fold (5) molar excess of wild-type oligonucleotide; lanes 6 and 7, same as lanes 4 and 5, but in the presence of mutated oligonucleotide. Bands I, II, and III indicate specific complexes, and NS indicates nonspecific bands. In the right figure, 2 closely migrating components of band I are shown. (B) Nuclear extracts from the control and homocysteine-stimulated cells were dephosphorylated in vitro as described in “Materials and methods.” Lane 1, probe and calf intestine alkaline phosphatase (CIAP); lanes 2 and 3, control extract without or with pretreatment by hexokinase and glucose, respectively; lanes 4 and 5, homocysteine-treated extract without and with hexokinase/glucose pretreatment, respectively; lane 6, homocysteine-treated extract with hexokinase/glucose pretreatment but without CIAP treatment. (C) Binding assay was performed in the presence of 0.1 to 0.2 μg of antibody specific for the indicated factors with the use of the control extract (lanes 1-8) and extract from the homocysteine-treated cells (lanes 9-16). Lanes 1 and 9, probe only; lanes 2 and 10, nuclear extract only; lanes 3 and 11, control IgG; lanes 4 and 12, anti-ATF2 antibody; lanes 5 and 13, anti-ATF3 antibody; lanes 6 and 14, anti-ATF4 antibody; lanes 7 and 15, anti–c-Jun antibody; lanes 8 and 16, probe with anti-ATF2 and anti–c-Jun antibody, respectively. Arrow indicates the supershifted band by anti-ATF2, anti-ATF3, and anti–c-Jun antibody.