In vitro kinase assays were performed using either purified effective PDK1 without first 52 amino acids or immunoprecipitated PDK1 from lysates of PC 3 cells. Cellular DNA synthesis, protein synthesis, and growth critiques For evaluation of DNA or protein synthesis, PC 3 cells were cultured in 24 well plates and treated with different concentrations of curcumin in FBS free MEM medium for the time. From then on 1 uCi/well of thymidine or L leucine were added into the cultures and incubated for 2 h. The cells were then mounted in one hundred thousand trichloroacetic acid at room-temperature for 15 min, and then washed twice with five full minutes TCA. The acid insoluble material was contained in 2 M NaOH immediately, and then aliquots were used to determine the radioactivity employing a liquid scintillation counter. For MTS cell proliferation assays, PC 3 cells were seeded in 96 well plates at a density of 5 103 cells/well, treated with different concentrations of curcumin for 24 h, then-20 Meristem ul of MTS reagent was added into each well and incubated for further 2 h. The density at 490 nm was read instantly using an uQuant microplate reader. Transient transfection and Western blotting Transient transfection was performed in line with the protocol provided by the company, and all experiments were performed 24 hrs after transfection. The cells as indicated were cultured in 6 well plates for 24 hrs accompanied by serum deprivation for 12 hrs, then treated with different concentrations of curcumin or substances in serum free media for the indicated time. After therapy, the cells were washed with cold PBS and harvested in 1X cell lysis buffer supplemented with protease inhibitor cocktail. Mobile lysates were centrifuged at 4 C, 13,000 natural product library g for 10 min, and the protein concentrations in supernatants were dependant on BCA protein assay. Aliquots of lysates each containing 30 ug of protein were boiled in 1x SDS loading buffer and resolved by 4 fifteen minutes SDS polyacrylamide gel electrophoresis. Proteins in gel were electro transferred to PVDF membrane using a semi-dry transport system. The membranes were blocked with five minutes fat free milk in phosphate buffered saline 0. Hands down the Tween 20 at room-temperature for 2 h, and then probed with specified major antibodies in 3% bovine serum albumin in PBST overnight at 4 C. Next the blots were washed with PBST for 10 min three times, and then incubated with matching HRPconjugated 2nd antibodies at room-temperature for 1 h. Then the blots were washed again in PBST for 10 min 3 x, and then were visualized by enhanced chemiluminiscence and scanned using a Gel Documentation 2,000 system. Actin was blotted for every test as loading get a grip on. COMPUTER 3 cells were cultured in 10 cm dishes and treated with the indicated concentrations of curcumin for 10 min, then washed and harvested in cell lysis buffer as described above.