The amplified fragments were separated on 1. 5% agarose gel and visualized with ethidium bromide Paclitaxel staining. Western blot analysis Inhibitors,Modulators,Libraries Protein samples were separated by SDS PAGE and blotted to nitrocellulose membrane. The membrane was incubated with antibody as specified, Inhibitors,Modulators,Libraries followed by secondary antibody conjugated with horseradish peroxidase. Specific antigen antibody complexes were detected by enhanced chemilumines cence. Western blot analysis was performed with the following antibodies anti Bax, anti caspase 3, anti PARP, anti Bcl 2, anti Akt, anti phospho Akt, anti caspase 8, anti caspase 9, anti DNA PKcs, anti DR5, anti caspase 10, anti pGSK 3b, anti GSK 3b, anti GSK 3a, anti c Myc, anti DR4 and anti b actin antibodies, Secondary antibodies were obtained from GE Healthcare.

Preparation Inhibitors,Modulators,Libraries of siRNA Transfection The siRNA used for targeted silencing of DNA PKcs were. CEM/VLB100 cells were transfected with 0. 1 uM siRNA for 48 h by oligofectamine according to the manufac tures protocol. In brief, CEM/VLB100 were seeded of 6 well Inhibitors,Modulators,Libraries plates and added to the siRNA/oligofectamine complex. Cells were incubated for 4 h at 37 C in serum free RPMI medium and then FBS was added. After 48 h, the cells were treated with TRAIL for another 24 h and col lected for western blot analysis to determine the levels of DNA PKcs and other indicated proteins. Apoptosis assay Cells were treated with or without TRAIL and/or indicated drug for 24 h and the cells were centrifuged and resuspended in 500 ul of the stain ing solution containing Annexin V fluorescein and propidium iodide in PBS.

After incubation at room temperature for 15 min, Inhibitors,Modulators,Libraries cells were analyzed by flow cytometry. Annexin V binds to those cells that express phosphatidyl serine on the outer layer customer review of the cell mem brane, and propidium iodide stains the cellular DNA of those cells that have a compromised cell membrane. This allows for the discrimination of live cells from apoptotic cells and necrotic cells. Flow cytometric dye efflux assay for multidrug resistance The accumulation of rhodamine 123, a fluorescent sub strate of P gp, in CEM and CEM/VLB100 cells treated with or without TRAIL was measured using a FACS flowcytometer equipped with an ultraviolet argon laser. Cell suspen sion from CEM and CEM/VLB100 cells treated with or without 10 ng/ml TRAIL for 6 h was incubated with rhodamine 123 at 37 C for 30 min. After incubation, the cells were washed with ice cold PBS and further incubated at 37 C for 3 h to allow P gp mediated drug efflux or on ice as control. Cells were pelleted by centrifugation at 500 g and resus pended in PBS containing.