Nitration of p190A induces RhoA activation in endothelial cells. (a) p190A was co-immunoprecipitated and precipitates were probed for phosphotyrosine (PY20) and p190A; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Human microvascular endothelial cells (dermal) overexpressing HA-tagged p190A, p190B, or p190A mutants Y1105F and Y1087F were treated with SIN-1. Nitration of endogenous p190A and exogenously expressed proteins was determined with 3-N antibody. Exogenous proteins were co-immunoprecipitated with anti-HA antibody; n = 3. (c) Live imaging of a genetically encoded FRET-based RhoA biosensor. Representative YFP and FRET/CFP ratio confocal images. Pixel intensities of ratio images were scaled from 0 to 5 and color-coded as indicated on the left. Bar, 10 µm. (d) FRET/CFP emission ratio; mean and SEM are as in Fig. 1 e; n = 13; *, P < 0.01. (e) RhoA-GTP was pulled down with Rhotekin-RBD beads. Resultant precipitates and 5% of cell extracts were probed for RhoA; results of two independent experiments are shown; n = 4. Molecular mass standards are indicated next to the gel blots in kilodaltons.