Ang-1 induces phosphorylation of eNOS on the inhibitory Thr⁴⁹⁷ site. (A) BAEC were stimulated with Ang-1 (100 ng/mL), VEGF (40 ng/mL), or both simultaneously for the indicated times. Cell lysates were probed for eNOS phosphorylation on Thr⁴⁹⁷ and Ser¹¹⁷⁹ using phospho-specific antibodies. Equal protein loading was confirmed by Western blotting for total eNOS. (B) Densitometric ratio of results presented in panel A of p-Thr⁴⁹⁷-eNOS (right panel) or p-Ser¹¹⁷⁹-eNOS (left panel) levels relative to total eNOS. Data were normalized with respect to control-unstimulated cells. Bar graph represents the average of 5 experiments expressed as mean ± SEM; *P < .05. (C) Tie2 counteracts the effects of VEGFR-2 on Thr⁴⁹⁷ residue of eNOS. Phosphorylation levels of eNOS on Thr⁴⁹⁷ were monitored in COS-7 cells transfected with eNOS plasmids in combination with expression vectors coding for VEGFR-2, Tie2, or both. Total cell lysates were analyzed for p-Thr⁴⁹⁷-eNOS and total eNOS. Bar graph shows the p-Thr⁴⁹⁷/total eNOS densitometric ratio expressed as mean ± SEM of 4 independent experiments; *P < .05 compared with phosphorylation levels of eNOS expressed with VEGFR-2. (D) Cellular localization of p-Thr⁴⁹⁷-eNOS in sparse (top panels) or confluent (bottom panels) BAEC after Ang-1 stimulation. BAEC were cultured on 0.1% gelatin-coated coverslips. Serum-starved cells were either untreated or treated with 100 ng/mL Ang-1 for 15 minutes. Cells were fixed, permeabilized, and incubated with anti–p-Thr⁴⁹⁷-eNOS (pAb; green) and anti-eNOS (mAb; red) primary antibodies. Immunofluorescent staining was performed as described in “Immunofluorescence” and observed by confocal microscopy (×63 objective).