Fig. 5

GSNOR but not Trx-1 is responsible for denitrosylation of VEGFD. A NCI-H1975 cells were transfected with Vector or Myc-GSNOR, cells were collected 48 h later, Western Blot was used to analyze VEGFD. B Western blot analyses in NCI-H1975 cells treated with the increasing concentrations of N91115 for 24 h. C NCI-H1975 cells were transfected with HA-VEGFD(WT) or HA-VEGFD(C277S), cultured for 24 h, and then treated with N91115 at 10 µM for further 24 h, Western Blot was used to analyze VEGFD. D Western blot analyses in NCI-H1975 cells transfected with vector, Flag-Trx-1(WT) or its inactive mutant Flag-Trx-1(CS) and then cultured for 24 h. E-F NCI-H1975 cells were transfected with HA-VEGFD(WT)/HA-VEGFD(C277S)/ HA-VEGFD(C215S)/ HA-VEGFD(C293S) in combination with Flag-Trx-1(WT)/Flag-Trx-1(CS), cell extract was harvested 48 h after transient transfection, Western Blot was used to analyze VEGFD. G-H Co-immunoprecipitation analyses and immunofluorescence staining in NCI-H1975 cells at 24 h post-transfection with HA-VEGFD(WT) and HA-VEGFD(C277S). H ABE and western blot analyses for detection of S-nitrosylated VEGFD and VEGFD in NCI-H1975 cells co-transfected with HA-VEGFD and GSNOR, cultured for 24 h, and then treated with N91115 at 10 µM for further 24 h. Data in (A), (B), (C), (D), (E), (F), (G), and (I) are representative of three independent experiments