FIGURE 1.

VEGF-mediated immediate and dynamic NFATc1 activation in primary cultured endothelial cells. A, schematic of spliced isoforms of human NFATc1 and the antigen for our generated mouse monoclonal antibody. A.A., amino acid. B, ChIP-Western blot analysis of immunoprecipitates using the anti-NFATc1 antibody from HUVEC extracts. The right column indicates the NFATc1 isoforms, n = 5., M.W. molecular weight. C, human NFATc1 (pCMV-NFATc1), human NFATc2 (pCMV-NFATc2), or mock control (pCMV) were transfected in COS7 cells. Whole cell extracts were prepared and blotted with an anti-NFATc1 or NFATc2 antibody. Anti-β-actin antibody was used as a loading control, representative of four independent experiments. D, NFAT immunofluorescent staining. NFAT localization in the nucleus versus the cytoplasm was calculated in HUVEC treated with VEGF at the indicated time points. Data are mean ± S.D. of the nuclear localization levels obtained from three independent experiments. *, p < 0.01 compared with without VEGF. E, immunofluorescent staining of NFATc1 (left column) in mock- and VEGF-treated HUVEC with or without CsA and MG-132. Merged images with PECAM1 and DAPI are shown in the right column. Scale bars = 50 μm. F, Western blot analyses with anti-NFATc1 antibody. HUVEC were treated with PBS (mock) or VEGF for 1 h in the presence or absence of CsA and MG-132. Subsequently, nuclear and cytosolic fractions were subjected to 10% SDS-PAGE. LaminA and Hsp90 are shown as nuclear and cytosolic markers, respectively. n = 3.