Fig. 3

VEGFD is S-nitrosylated. A GSNO was administered to eight-week-old C57/BL6 female mice at the doses of 0.5 and 1.5 mg/kg (blank control) by single intratracheal instillation, RNA and Proteins were isolated from lungs. mRNA level and quantification of VEGFD protein level using Western Blot analysis (n = 3 per group). B-C NCI-H1975 cells treated with GSNO or L-NAME. qRT-PCR, quantification of WB analysis for VEGFD. Numerical data were expressed as mean ± SD (each n = 3). D HEK-293T cells were transfected with HA-VEGFD, cell extract was harvested 48 h after transient transfection. Thermo Scientific™ Pierce™ S-Nitrosylation Western Blot Kit detects the S-nitrosylated VEGFD. E-F HEK-293T cells transfected with vector or VEGFD-expressing construct, cultured for 24 h, and then treated with DTT (10µM) or GSNO (50µM) for further 24 h, Thermo Scientific™ Pierce™ S-Nitrosylation Western Blot Kit detects the S-nitrosylated VEGFD in cell lysate. Data in (D), (E), and (F) are representative of three independent experiments, and data in (A), (B), and (C) represent the mean ± SEM of triplicate samples. *P < 0.05, **P < 0.01, Student’s t test