Figure 4.

NOSTRIN decreases NO levels in endothelial cells without affecting the phopho-eNOS levels, and the effect of NOSTRIN on endothelial cell function-associated proteins is eNOS-independent. A, NO production was measured in culture supernatants in the form of nitrites. Error bars represent S.E. from three different biological replicates. B, Western blotting analysis of eNOS and phospho-eNOS from endothelial cells from the four treatment groups. C, relative expression of phospho-eNOS with respect to eNOS was quantified by ImageJ software after normalization with rpL7 using three biological replicates from B. D, Western blotting analysis of protein levels of 14 of 19 genes, associated with various endothelial cell functions were reproducibly altered by NOSTRIN overexpression in three biological replicates. The positions of the molecular-weight markers for the spliced blots are shown on the left side of the image. E, quantification by ImageJ software of the proteins relative to rpL7 using three biological replicates from D. F–H, ELISA analysis of secreted cytokines from endothelial cells transfected with NOSTRIN and vector backbone (Control). Cytokines were quantified in pg relative to total protein estimated in mg. Each experiment was repeated three times with different biological samples. Error bars represent S.E. *, p < 0.05; **, p < 0.01; and ***, p < 0.005.