and transferred to membranes (Hybond-ECL, Amersham Biosci- ences). Membranes were blocked in either 5% milk or 5% BSA in PBS-Tween (0.05%) and incubated with mouse anti-Rac1 (Clone 23A8, Upstate), mouse anti-Cdc42 (Chemicon), mouse anti-RhoA (Santa Cruz) or rabbit anti-VEGFR2 (Cell Signalling Technolo- gies) antibodies. Membranes were washed three times in PBS- Tween (0.05%) and subsequently incubated with HRP-conjugated IgGs. Immunoreactive bands were visualised by incubating the membranes with ECL chemiluminescence reagents (Amersham Life Science) and exposing the membranes to autoradiography film (Amersham Life Science). After detection of the protein of interest, membranes were stripped with stripping solution (Chemicon), and re-blotted with mouse anti-Hsc-70 antibody. Densitometry was performed using a gel acquisition and analysis set-up (UV Products, Ltd.). Band densities were normalised to Hsc-70 to make quantitative measurements of the protein of interest. Rho GTPases activity assays A construct encoding glutathione S-transferase (GST) linked to the amino acid residues 57 to 141 of the PAK-CRIB domain (GST-PAK-CRIB; kindly provided by Dr. John Collard, King’s College, London, UK and Dr. Vania Braga, Imperial College, London, UK), was used to pull down active Rac1 and Cdc42 from MLEC cell lysates transfected with scrambled or Rac1 siRNA. Pull-downs were performed as described [66]. Samples of lysate preincubation (total protein) and postincubation with the GST- PAK-CRIB beads (active protein) were resolved in 12% polyacrylamide gels, transferred to PVDF membranes, and immunoblotted with anti-Rac1 or anti-Cdc42 antibodies as described above. The proportion of active Rac1 and Cdc42 relative to their the total amount in the cells was determined by densitometry (UV Products, Ltd.). Active levels of RhoA in MLEC transfected with scrambled or Rac1 siRNA, were assessed using the G-LISATM RhoA activation assay Biochem KitTM (Cystoske- leton) following the manufacturer’s instructions. The degree of RhoA activation was determined by comparing absorbance readings at 490 nm from Rho control protein (constitutively active RhoA protein) samples versus cell lysates of interest. In vivo tumorigenesis and endothelial-specific Rac1- deletion Rac1 flox/flox and Rac1 flox/flox PDGFB-iCreER mice were implanted subcutaneously with tamoxifen (Rac1 flox/flox and Rac1 flox/flox PDGFB-iCreER) or placebo (Rac1 flox/flox PDGFB-iCreER) 1 mg-slow release pellets in the scruff of the neck. One day later B16F0 tumor cells (16106) were injected subcutaneously in the flank. At day 10 after tumor cell injections, tumors were dissected out and processed for histology. In vivo VEGF-induced angiogenesis assays Mouse subcutaneous sponge angiogenesis assays [67] were carried out by implanting two autoclaved sponges subcutaneously into the flanks of wild-type/Tie1-Cre+ and b3-null/Tie1-Cre+ mice. Sponges were injected in situ as follows: on days 1, 5 and 9 with 100 ml of 10 ng/ml VEGF-A164; on days 3, 7 and 13 with pSico-Con, pSico-Rac1 and pSico-Flk-1 (106 i.u/ml) in 100 ml of 10 ng/ml VEGF-A164. At day 15 after sponge implantations, the mice were euthanized by CO2 asphyxiation, sponges excised and fixed for paraffin embedding. Additionally growth factor induced angiogenesis assays were carried out by implanting two autoclaved sponges subcutaneously into the flanks of wild-type and b3-null mice. These sponges were further injected in situ every 2 days with Rac1 and Tumor Angiogenesis Con siRNA (3 mg) in 100 ml of PBS alone, Con siRNA (3 mg) or Rac1 specific-siRNA (3 mg) in 100 ml of PBS containing 10 ng/ml VEGF-A164. At day 15 after sponge implantation, the mice were euthanized by CO2 asphyxiation, sponges excised and processed for histology. Quantification of blood vessel density Tumor and sponge samples were snap frozen for cryosectioning or fixed in 4% paraformaldehyde for paraffin-embedding, respectively. Frozen sections were brought to room temperature, fixed in 4% PFA and rehydrated in PBS, whilst paraffin embedded sections were deparaffinized, rehydrated in PBS and microwaved on full power (950 W) in sodium citrate buffer pH 6.0, before incubation with rat anti-mouse PECAM-1 (Pharmingen), rabbit anti-green fluorescent protein (GFP) (Mol Probes) and rat anti- mouse endomucin (kindly provided by Dr. Dietmar Vestweber, Max-Planck-Institute of Molecular Biomedicine, Muenster, Ger- many) followed by incubation with goat anti-rat Alexa Fluor-546, goat anti-rabbit Alexa Fluor-488 (both from Mol Probes) and biotinylated rabbit anti-rat antibody (Vector Laboratories), respectively. When biotinylated antibody was used an alkaline phosphatase-based system (VECTASTAIN ABC-AP, Vector Laboratories, Peterborough, UK) with Vector Blue substrate kit (Vector Laboratories, Peterborough, UK) was used to amplify the signal according to the manufacturer’s instructions. Blood vessel density in immunofluorescent stained sections from size-matched tumors was determined by counting the number of PECAM-1+/ GFP2 or PECAM-1+ blood vessels present across an entire section and then dividing by the total area of the section. Blood vessel density in immuno alkaline-phosphatase stained sections from sponges was determined by counting the number of endomucin+/ GFP2 stained blood vessels present across an entire section and then dividing by the total area of the section. In all cases blood vessel counting was performed in a double-blind fashion using an epifluorescence microscope Zeiss (Carl Zeiss, Jena, Germany) with associated digital camera (Hamamatsu Photonics, Ltd., Welwyn Garden City, UK) and using Open Lab software v4.1software (Improvission, Coventry, UK) to capture images and calculate areas of the sections. Ex vivo mouse aortic ring assays Wild-type and b3-integrin-deficient mice were sacrificed by cervical dislocation, thoracic aortas removed under sterile conditions, periaortic fibroadipose tissue was removed and aortas were cut into 0.5–1 mm-wide rings. After 24 hours siRNA transfected aortic rings were rinsed in D-MEM supplemented with 6 mM L-glutamine and 2% FCS (basal medium) and each ring was transferred to a well of a 96-well plate containing polymerized growth factor reduced (GFR)- Matrigel (Becton Dickson, Beds, UK) before adding 150 ml of basal medium. The medium was supplemented with 25 ng/ml VEGF-A164 and DC101 at 20 mg/ml [27]. Where DC101 was used, the GFR-Matrigel was supplemented with 20 mg/ml DC101 antibody just before polymerisation. The cultures were grown at 37uC and 8% CO2, for up to 8 days. Aortic rings were examined microscopically and media including VEGF and DC101 was changed every 2 days. On day 5, phase contrast photomicrographs were taken using an inverted microscope (Carl Zeiss) equipped with a camera (Hamamatsu Photonics, Ltd.) and angiogenesis calculated by counting the number of capillary sprouts per ring. Rac1 expression in transfected aortic rings was assessed by using both semiquantitative RT-PCR and Western blot analysis. PLoS ONE | www.plosone.org 15 March 2010 | Volume 5 | Issue 3 | e9766