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Fig. 3
Figure 3. DDAH1 mediates acute and chronic VEGF-mediated signaling pathways in ECs (A) HUVECs were infected with lentivirus expressing of control shRNA and DDAH1 shRNA, cultured in low-serum EMB for 4 h and then exposed to stimuli of acute (single stimulus of VEGF at 10 ng/mL) and chronic VEGF (three stimuli of VEGF at 10 ng/mL with 30 min intervals), respectively. Proteins were collected 10 min after VEGF stimulation and analyzed with western blotting (A). (B) Control and DDAH1 knockdown HUVECs were labeled with DAF-FM (2.5 μM) and then exposed to acute and chronic VEGF stimulation, respectively. DAF-FM fluorescence was measured at Excitation/Emission of 495/515 nm. N numbers are presented in the scatter plot. Data is expressed as mean ± SD. ∗∗∗, p < 0.001. (C) HUVECs were infected with retrovirus expressing of LacZ and DDAH1, cultured in low-serum EMB and then exposed to acute and chronic VEGF stimulation. Proteins were collected 10 min after VEGF stimulation and analyzed with western blotting.