Effects of L-NAME treatment on eNOS, VEGF and KDR expression.

<p>(A) Densitometric analysis of eNOS protein expression. ***p<0.001; <i>t</i> test; n = 11. Inset: a representative blot out of eleven is shown. Total eNOS protein was evaluated by western blotting on lysates prepared from control cells (lane 1) or from 48 h L-NAME treated cells (lane 2). β-actin was used as a loading control. (B) eNOS RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. No significant differences between control and L-NAME treated cells (<i>t</i> test, n = 3). (C) VEGF and KDR RNA levels were measured by RT-qPCR and normalized to the level of the housekeeping gene 18S. **p<0.01 <i>vs</i> control cells (CTRL); <i>t</i> test; n = 6−4 for VEGF and KDR, respectively. (D) VEGF protein levels were detected by ELISA measurement in conditioned media collected from control or 48 h L-NAME treated cells. Results are expressed as pg of VEGF normalized to the cell protein content (pg/mg protein). **p<0.01; <i>t</i> test; n = 3. (E) KDR protein was visualized by western blot after immunoprecipitation with KDR antibodies of HUVEC lysates obtained from control (lane 1) or from 48 h L-NAME treated cells (lane 2). An aliquot of total cell lysates was immunoblotted with β-actin antibodies as a control (input). Shown is a representative blot of 2 comparable experiments. (F) Control cells (lanes 1 and 2) or 48 h L-NAME treated cells (lanes 3 and 4) were stimulated for 5 min with 25 ng/ml VEGF. Aliquots of cell lysates were separated by 10% SDS-PAGE and immunoblotted with the indicated antibodies. Actin was used as a loading control. Shown is a representative blot of 4 comparable experiments.