5% FBS for 12 h. After being washed with fresh medium, cells were treated with santalol for 30 min, followed by the stim ulation with 50 ng/mL of VEGF for 2 min or 20 min for mTOR pathway kinase ac tivation or 20 min for ERK inhibitor 17-DMAG pathway phosphorylation. To examine mTOR pathway in prostate tumor cells, normal cultured PC 3 or LNCaP cells were directly Inhibitors,Modulators,Libraries treated with indicated dilutions of santalol for 6 h. The whole cell ex tracts were prepared in RIPA buffer supplemented with PMSF and proteinase inhibitor cocktail before use. Pro teins are resolved by electrophoresis then transferred out of the SDS PAGE gel and onto polyvinylidene difluoride membranes.

The Inhibitors,Modulators,Libraries membranes were incubated with primary antibodies anti B actin, anti VEGFR2, anti AKT, anti ERK1/2, anti mTOR, anti S6K, anti Src, anti FAK, phospho specific anti VEGFR2, anti VEGFR2, antiAKT, anti ERK1/2, anti mTOR, anti S6K, anti Src and anti FAK followed by the addition of sec ondary antibodies conjugated to horserad ish peroxidase. Anti cleaved caspase 3 was used for detecting apoptosis. Poly polymerase cleav age was detected by anti poly polymerase antibody. Proteins bands were visualized using Phototope HRP Western blotting detection System according to the manufacturers protocol. For tumor sections, radio immunoprecipitation assay buffer was added to the sections and homogenized with electric homogenizer. After incubation for 20 minutes on ice, samples were cen trifuged for 30 minutes at 12,000 rpm at 4 C and super natant was collected as total cell lysate. SDS PAGE was carried out as described previously.

Enzyme linked immunosorbent assay The levels of VEGF were determined by VEGF ELISA kit according to the manufacturers Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries instruction. Flow cytometry fluorescence activated cell sorting analysis About 2 106 HUVEC or PC3 or LNCaP cells were treated with santalol at 37 C, 5% CO2 incubator for 24 h. The cells were collected and analyzed in a FACS Vantage SE DiVa flow cytometer with propidium iodide staining. The cell population percent ages at sub G1 were defined as apoptotic cell percentages. Hoechst staining About 2 106 HUVEC or PC3 cells were seeded on 8 well chamber slides and grown to sub confluence. After treatments for 14 h with the indicated concentrations of santalol in complete medium, cells were washed and fixed. Chamber slides were stained with Hoechst, mounted, and observed under a fluorescence microscope.

Inhibitors,Modulators,Libraries The percentage of control and santalol treated cells showing chromatin condensation was eval uated in ten vision fields from two independent experi ments. Cytometric selleck inhibitor bead array analysis for active caspase 3 BD Human Active Caspase 3 CBA Kit was used to quantify active caspase 3 levels following manufacturers protocol. Rat aortic ring assay The rat aortic ring assay was used as an ex vivo angio genesis study model. Dorsal aorta from a freshly sacrificed Sprague Dawley rat was taken out in a sterile manner and rinsed in ice cold PBS.