Figure 3.

Ephrin-A1-induced NFAT1 activation is EphA2-dependent. A) Treatment with siRNA directed against EphA2 significantly blunted EphA2 expression in HAECs after 48 hours; n = 4. B) Treatment with EphA4 siRNA significantly blunted EphA4 expression in HAECs after 48 hours; n=4. C) Ephrin-A1-induced NFAT nuclear translocation in endothelial cells transfected with siRNA directed against EphA2 was assessed by Western blotting for NFAT1 in the nuclear fraction, n = 4. D & E) Ephrin-A1-induced NFAT nuclear translocation in endothelial cells transfected with siRNA directed against EphA2 or EphA4 was assessed by immunocytochemistry for NFAT1. D) Representative images are shown. E) Nuclear translocation was quantified by scoring cells for positive nuclear NFAT1 staining. At least 100 cells were assessed for each condition per experiment. n = 4. F & G) HAECs were treated with the EphA2 kinase inhibitor ALW-II-41–27 (indicated doses) and stimulated with dimerized ephrin-A1-Fc (2 μg/mL) for 15 minutes to induce NFAT activation. NFAT nuclear translocation was measured by (F) subcellular fractionation and Western blotting and (G) by immunocytochemistry and nuclear scoring. Representative images are shown, and at least 100 cells were scored for NFAT nuclear translocation for each condition per experiment. n = 4. Statistical comparisons were made with either Student’s T-test (A/B), 2-way ANOVA with Bonferroni posttest (C,E,F) or 1-way ANOVA with Bonferroni posttest (G).