Fig 5. Endothelial NOS deficiency prevents LPS mediated RhoA activation and nitration at Y³⁴ in the mouse lung.

Immunoblot analysis of lung tissue extracts indicated no differences in RhoA protein levels in either wild-type or eNOS^-/- mice in the absence or presence of LPS (A). However, LPS induced a significant increase in RhoA activity in the lungs of wild-type mice but not in eNOS^-/- mice (B). Recombinant RhoA protein (30 μg) incubated in the presence or absence of SIN-1 (25 mM, 1 h at 37°C) was immunoblotted with an antibody raised against nitro-Y³⁴ RhoA and then normalized with total RhoA antibody. The nitro-Y³⁴ RhoA antibody preferentially bound to nitrated RhoA (C). RhoA Y³⁴ nitration levels in lung extracts were significantly elevated in LPS exposed wild-type mice; however, LPS did not alter RhoA Y³⁴ nitration in the lungs of eNOS^-/- mice (D). Values are mean ± SEM, n = 6–10. *p<0.05 vs. Wild-type+Vehicle, †P<0.05 vs. Wild-type+LPS.