Despite numerous reports implicating NADPH oxidases (Nox) in the pathogenesis of 
many diseases, precise regulation of this family of professional reactive oxygen 
species (ROS) producers remains unclear. A unique member of this family, Nox1 
oxidase, functions as either a canonical or hybrid system using Nox organizing 
subunit 1 (NoxO1) or p47(phox), respectively, the latter of which is functional 
in vascular smooth muscle cells (VSMC). In this manuscript, we identify critical 
requirement of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50; aka 
NHERF1) for Nox1 activation and downstream responses. Superoxide (O2 (•-)) 
production induced by angiotensin II (AngII) was absent in mouse EBP50 KO VSMC 
vs. WT. Moreover, ex vivo incubation of aortas with AngII showed a significant 
increase in O2 (•-) in WT but not EBP50 or Nox1 nulls. Similarly, 
lipopolysaccharide (LPS)-induced oxidative stress was attenuated in femoral 
arteries from EBP50 KO vs. WT. In silico analyses confirmed by confocal 
microscopy, immunoprecipitation, proximity ligation assay, FRET, and 
gain-/loss-of-function mutagenesis revealed binding of EBP50, via its PDZ 
domains, to a specific motif in p47(phox) Functional studies revealed 
AngII-induced hypertrophy was absent in EBP50 KOs, and in VSMC overexpressing 
EBP50, Nox1 gene silencing abolished VSMC hypertrophy. Finally, ex vivo 
measurement of lumen diameter in mouse resistance arteries exhibited attenuated 
AngII-induced vasoconstriction in EBP50 KO vs. WT. Taken together, our data 
identify EBP50 as a previously unidentified regulator of Nox1 and support that 
it promotes Nox1 activity by binding p47(phox) This interaction is pivotal for 
agonist-induced smooth muscle ROS, hypertrophy, and vasoconstriction and has 
implications for ROS-mediated physiological and pathophysiological processes.