Real time RT PCR was carried out in a Rotorgene 6000 using SYBR Green Jump Start Taq ReadyMix and cDNA corresponding to 25 ng RNA. Cycling conditions inhibitor Rapamycin were 2 minutes initial Inhibitors,Modulators,Libraries incubation at 50 C, followed by 5 min utes at 95 C and amplification for 40 cycles at 95 C for 15 seconds, 60 C for 30 seconds and 70 C for 40 seconds. The cycle threshold value of each reaction was determined using Corbetts Rotor Gene 6 software. To confirm primer specificity, amplicon melting curves were recorded after cycle 40 by heating from 60 to 95 C with a ramp speed of 0. 5 C every second. Gene specific mouse cDNA sequences were obtained from GenBank, and primers were designed using the Primer3 design package hosted by the Whitehead Institute for Biomedi cal Research.

Inhibitors,Modulators,Libraries Primers were designed to lie in different exons to prevent the amplification of genomic DNA, and optimised to work at an annealing tempera ture of 60 C. Primer amplification efficiencies were veri fied by real time RT PCR on serial dilutions of cDNA according to Pfaffl. The expression of target genes was expressed as mRNA level in fold change relative to the mRNA level in tissue of control mice after normalis ing to the Arp gene using the comparative Ct method. Mouse Angiogenesis RT2 Profiler PCR Array The Mouse Angiogenesis RT2 Profiler PCR Array profiles the expression of 84 genes involved in angiogenesis as well as five housekeeping genes by real time PCR using the Inhibitors,Modulators,Libraries SYBR Green detection method. Therefore, 500 ng of total RNA was reverse transcribed using the Reaction Ready First Strand cDNA Synthesis Kit following manufacturers instruc tions.

The generated cDNA was diluted with an appropri ate volume of instrument specific 2x SuperArray RT2 Real Time SYBR Green PCR Master Mix and ultra pure water, and 25 ��l of this reaction mix was added to each well of the PCR array. The real time PCR reaction was performed in an ABI 7700 thermocycler Inhibitors,Modulators,Libraries applying the following program, 2 minutes at 50 C, 10 minutes at 95 C and 40 cycles of 15 seconds at 95 C and 1 minute at 60 C. The ABI PRISM 7700 Sequence Detection System was used to calculate the Ct value for each well. Data were nor malised to four housekeeping genes and analysed by the comparative Ct method. Preparation of paw homogenates Mouse paws were skinned, snap frozen, pulverised, and homogenised in 1. 5 ml ice cold sterile PBS containing protease inhibitor cocktail.

Debris were removed by centrifugation at 4 C 13. 000 rpm for 15 minutes and the supernatants were filtered using a 0. 2 ��m sterile syringe filter. The protein content of the homogenates was determined using the Pierce BCA protein assay kit following manufacturers guidelines. Inhibitors,Modulators,Libraries Endothelial cell migration assay Human microvascular endothelial cells were cultured in MCDB131 selleck bio medium containing 10% foetal calf serum, 100U ml penicillin and 0.