Antibodies distinct for phospho Akt and phospho IKK IKK were ordered from New England Biolabs. The pcDNA was generously provided by Dr. M. C. Chen. PBK CMV Lac Z and pgl2 ELAM Luc were generously supplied by Dr. T. W. Lin. Lenalidomide clinical trial The PGE2 enzyme immunoassay kit was obtained form Cayman. ATP was obtained fromAmersham Pharmacia Biotech. The myc His tagged expression construct for that dominant negative Akt1 K179M mutant was a kind gift from Prof. C. M. Teng. Gene PORTERTM 2 was obtained from Gene Therapy System. All components for sodium dodecylsulfate polyacrylamide gel electrophoresis were purchased from Bio Rad. All the substances were obtained from Sigma. The mouse macrophage cell line, RAW264. 7,was purchased from American Type Culture Collection, and cellswere maintained in DMEM/Hams F 12 nutrient mixture containing 10% FCS, 100 U/ml of penicillin G, and 100 g/ml streptomycin in a humidified 37 C incubator. After reaching confluence, cells were seeded onto either 6 cm dishes for immunoblotting, kinase assays, the Rac activity assay, and company immunoprecipitation, or 12 well plates for transfection, Endosymbiotic theory PGE2 launch, and B luciferase assays. B and transfection luciferase assays For these assays, 2 105 RAW 264. 7 cells were seeded onto 12 well plates and cells were transfected the next day using GenePORTERTM 2 with 0. 5 g of pGL2 ELAM Luc and 0. 5 g of pBKCMVLac Z. After 24 h, the medium was aspirated and replaced with new DMEM/Hams F12 containing 10% FBS, and then activated with car or PGN for another 24 h before being gathered. To assess the effects of PI3K and Akt inhibitors, drugs were added to cells 20 min before PGN improvement. To assay the effects of AktDN and RacN17, cells were cotransfected with RacN17 or AktDN, pGL2 ELAM Luc, and pBK CMV Lac Z for 24 h and then treated with Ibrutinib structure PGN. Luciferase activitywas determined using a luciferase assay system, and was normalized on the basis of Lac Z expression. The amount of induction of luciferase activity was compared as a ratio of cells with and without stimulation. Cellswere transfected with 1 g of pEGFP, a green fluorescence protein expression vector for 24 h, to find out the transfection efficiency. After treatment, the medium was aspirated and replaced with fresh DMEM/Hams F12 containing 10% FBS for another 24 h. Cells were discovered under ugly laser scanning confocal microscopy. The transfection efficacy was thought as the proportion of cells expressing GFP. The price of GFP was about 50%. To look for the words of Akt, tubulin, phosphoAkt, COX 2, phospho IKK /IKK, IKK, phospho p65, and p65 in RAW 264. 7 macrophages, proteins were extracted and a Western blot analysis was performed as described previously.