Nitric Oxide Synthase Trafficker (NOSTRIN) is a scaffolding protein that regulates the subcellular localization and activity of endothelial nitric oxide synthase (eNOS), a key enzyme in vascular nitric oxide (NO) production. NOSTRIN facilitates eNOS trafficking to caveolae, specialized membrane microdomains, where it modulates NO signaling. Dysregulation of NOSTRIN has been linked to vascular disorders, including preeclampsia, where its overexpression correlates with reduced eNOS activity and impaired vasodilation. NOSTRIN interacts with Caveolin 1 (CAV1), a structural protein of caveolae, through direct binding, influencing eNOS sequestration and NO bioavailability.To detect the activity of recombinant NOSTRIN, a functional ELISA assay was performed to evaluate the interaction between recombinant human NOSTRIN and recombinant human CAV1.Briefly,
NOSTRIN was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to CAV1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-NOSTRIN pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant rat NOSTRIN and recombinant human CAV1 was shown in Figure 1, the EC50 for this effect is 46.38 µg/mL.