Jurkat T cells were cultured in RPMI1640 containing 10% FBS. For PLC assays and co immunoprecipitation assays, HEK293 cells were seeded at 60% confluency into 12 well plates or 6 well plates, respectively. Transfection was performed on the follow ing day using Lipofectamine PLUSTM reagents. For the establishment of selleck chemical EPZ-5676 stable cell lines, exponentially growing HEK293 cells were transfected with cDNA of BK2R, B2AR or fMLPR in pcDNA3. 1 zeo using Lipofectamine PLUSTM. The cells were then selected with Zeocin. 293fMLPR G16 cells were established by transient transfection of 293fMLPR stable cell lines with G16 in pcDNA3. In vitro PKD Assay Twenty four hours after transfection, HEK293 cells were serum starved overnight and then treated with 500 ul of ice cold detergent containing lysis buffer.

Lysates obtained were subjected to in vitro PKD kinase assay. Fifty ul Inhibitors,Modulators,Libraries of each supernatant was used for the detection of PKD isoform expression and stimu latory phosphorylation, and the remaining lysate was incubated overnight at 4 C with specific affinity gels to immune precipitate the corresponding PKD isoform. The resulting immuno precipitates Inhibitors,Modulators,Libraries were washed twice with lysis buffer and twice with kinase assay buffer. Washed immunoprecipitates were resuspended in 40 ul of kinase assay buffer containing 2. 5 mgml of Syntide 2, and the kinase reactions were initiated by the addition of 10 ul of ATP buffer containing 1 uCi of ATP per sample. After 10 min incubation Inhibitors,Modulators,Libraries at 30 C with occasional shaking, the reactions were termi nated by adding 100 ul of 75 mM H3PO4 and spotting 75 ul of the reaction mix onto P 81 phosphocellulose paper.

Free ATP was separated from the labelled substrate by washing the P 81 Inhibitors,Modulators,Libraries paper four times in 75 mM H3PO4. The papers were dried and the radioactivity incorporated into Syntide 2 was determined by scintillation counting. Electroporation The knock down of PKD1, PKD2 and PKD3 was performed by introducing the Inhibitors,Modulators,Libraries corresponding PKD isoform specific siRNA from Invitrogen using NucleofectorW Kit V from Lonza. Briefly, 1��106 cells per sample were resuspended in NucleofectorW Solution and supplement provided at room temperature. siRNA against PKD1, PKD2 or PKD3 was added to the sam ples and then electroporated using the NucleofectorW. Electroporated cells were then incubated at room temperature for 10 min before transferring them into the 12 well Axitinib clinical plate with culture medium. The knock down of PLCB1, PLCB2 and PLCB3 was performed in similar manner, with the corresponding isoform specific siRNA obtained from Santa Cruz Biotechnology. Western blotting analysis Cells in 12 well plate were lysed in 300 ul of ice cold lysis buffer. Clarified lysates were resolved on 1 u2% SDS polyacrylamide gels and then transferred to nitrocellulose membranes.