Figure 2.

TSP1 inhibits agonist-stimulated phosphorylation of eNOS. HUVEC were serum starved and pre-treated with TSP1 (2.2 nmol/L 15 min) followed by addition of ionomycin (3 µmol/L) (A) or ACh (10 µmol/L) for the times indicated (B). Lysates were prepared and western blot analysis of eNOS phosphorylation at serine¹¹⁷⁷ determined. A representative blot of three experiments for each treatment is shown. Arterial segments from wild-type mice were pre-treated with TSP1 (2.2 nmol/L 15 min) followed by ACh (10 µmol/L) ± and lysates blotted against eNOS phosphorylation at serine¹¹⁷⁷ (C). A representative blot of three separate experiments is presented. HUVEC were serum starved and pre-treated with TSP1 (2.2 nmol/L 15 min) followed by ACh (10 µmol/L) for the indicated times. eNOS was immunoprecipitated and then western blotted against Hsp90 (D). A representative blot of three separate experiments is presented. For western blots, expression was quantified by densitometry calculated by measuring intensity of bands using Image J and normalized to either β-actin or eNOS expression.