FIGURE 7.

Imaging of generation and immunostaining in BAECs. A, confocal microscopy measurements of generation from uncoupled eNOS in endothelial cells. BH₄ depletion was achieved by incubation of BAECs with 5 mm 2,4-diamino-6-hydroxypyrimidine (DAHP) 18 h at 37 °C. The generation was visualized from BH₄-depleted BAECs loaded with DHE that reacts to form HE, that exhibits red fluorescence. The blue color corresponds to the nuclei stained with DAPI. Measurable fluorescence was seen only when the BH₄-depleted cells were treated with CaI, VEGF, or PMA, and this was inhibited by l-NAME. B, immunostaining using phosphorylation specific anti-eNOS antibodies in BH₄-depleted BAECs. The left column shows the results using the anti-phospho-Ser-1179 antibody. Ser-1179 phosphorylation was detected in BH₄-depleted BAECs when the cells were treated with CaI or VEGF but not with PMA. The right column shows the results using the anti-phospho-Thr-497 antibody, demonstrating that PMA treatment produced strong phosphorylation of Thr-497. These two immunostaining experiments were done in two sets of cells, both under the same conditions.