Elevated ras signaling and increased Nfatc1 nuclear localization in hearts of Nf1−/− embryos. a–d, Activated MAPK (arrows) detected by immunohistochemistsry in sections of outflow tract (a,b) and atrioventricular (c,d) endocardial cushions from E11.5 wild-type (a,c) and Nf1−/− (b,d) embryos. e, Higher levels of activated ras were present in Nf1ec embryos as detected by pull-down assay with the ras binding domain of raf. wt, wild-type. f,g, Nfatc1 was localized to the cytoplasm of endothelial cells from E9.5 wild-type (yellow arrow, f) embryos and to the nucleus in Nf1−/− littermates (yellow arrow, g). h,i, Nfatc1 was in the cytoplasm of endothelial cells cultured from wild-type endocardial-cushion explants infected with adenovirus expressing lacZ (h) but in the nucleus of cells infected with rasV12 virus (i). j,k, Human umbilical vein endothelial cells were transfected with Nfatc1 alone (j) or Nfatc1 plus rasV12 (k), which resulted in nuclear localization of Nfatc1 (arrow, k).