Anti rac1

Neutrophils were lysed and centrifuged at 14,000 g for 10 min at 4°C. The supernatants were incubated with the GST-PBD (p21-binding domain of PAK1) beads for Rac1 and Cdc42 activity assays or GST-RBD (Rho-binding domain of Rhotekin) for RhoA activity assay following the manufacturer’s instructions. Levels of bead-bound GTP-Rac1/Cdc42/RhoA and total Rac1/Cdc42/RhoA were analyzed by immunoblot with antibodies as follows: anti-Rac1 (1:1,000; Abcam), anti-Cdc42 (1:1,000; Abcam), anti-RhoA (1:1,000; Abcam).

Vessels were solubilized in lysis buffer containing 20 mmol/L Tris‐HCl, 150 mmol/L NaCl, 20 mmol/L NaF, 2 mmol/L sodium orthovanadate, 1% Nonidet, 100 μg/mL leupeptin, 100 μg/mL aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride. Then, samples were left on ice for 30 minutes and centrifuged at 10 621g for 20 minutes, and the supernatants were used to perform immunoblot analysis. Total protein levels were determined using the Bradford method. Rac1 activity was determined using a commercially available kit (Cell BioLabs Inc, San Diego, CA, STA‐401‐1) as described below. Forty micrograms of proteins were resolved on 10% SDS‐PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti‐Rac1‐GTP (1:1000, Cell BioLabs) or anti‐Rac1 (1:1000, Abcam, Cambridge, UK); with anti–RhoA‐associated kinase 1 (ROCK1) (1:1000 (abcam); with anti‐p–endothelial nitric oxide synthase (eNOS) phosphorylated on serine 1177 (1:800, Abcam) or anti‐total‐eNOS (1:800, Abcam); or with anti‐β‐actin (1:1000, Cell Signaling, Danvers, MA). Horseradish‐peroxidase–conjugated secondary antibodies were used at 1:3000 dilution (Bio‐Rad Laboratories, Hercules, CA). Protein bands were detected by ECL Prime (Amersham Biosciences, Little Chalfont, UK), and densitometry analysis was performed using Quantity One software (Bio‐Rad Laboratories).