NF kB Reporter Assays Cells were transfected with 3X NF kB luciferase reporter and pcDNA3. 2N NaOH, and analyzed on a scintillation counter. Cells were plated in 100 mm dishes, restored with media containing drugs the following day when cells were 400-word confluent, and harvested after 72 h. Cells were labeled with bromodeoxyuridine for 1 h at 37uC, trypsinized, permeabilized and washed with ethanol, stained with fluorescein isothiocyanate conjugated AG-1478 price anti BrdU antibody and propidium iodide, and analyzed by fluorescence activated cell sorting using Cell Quest computer software and Modfit research. Apoptosis Assays PARP and caspase 3 cleavage. Cells were treated with drugs the following day, and plated in 60 mm dishes. After 40 h, attached and detached cells were lysed in RIPA buffer, and blots were incubated with GAPDH antibodies and PARP, caspase 3. Fluorescent Caspase 3/7 assay. Cells were plated in 6 well meals in triplicate, drug addressed the following day when cells were 401(k) confluent, and attached and detached cells were lysed 40 h later. Lysate was incubated with substrate, and fluorescence recognized at 360 nm/460 nm with a Synergy Resonance (chemistry) 2 microplate reader. When cells were 401(k) confluent cells were plated in 100 mm dishes, and treated with drugs the following morning. After 40 h, cells were trypsinized, washed in DMEM /5% FBS, counted, and cells were incubated with Annexin V APC and propidium iodide for 159 ahead of FACS analysis. Doxorubicin Accumulation Assays Subconfluent cells were incubated with either rhodamine 123 or doxorubicin in the presence of verapamil or imatinib for 309, washed extensively, incubated with verapamil or imatinib for yet another 459, and analyzed by FACS to evaluate rhodamine 123 or doxorubicin intracellular storage. 1 plasmids, picked with G418, and clones were pooled. Cells stably expressing the reporter were plated in triplicate, addressed with doxorubicin, imatinib or the mixture for 24 h, lysed purchase Imatinib in lysis buffer, lysate was incubated with luciferin for 20, and luminescence resulting from activity measured with the Synergy 2 microplate reader. Nuclear fractionation assays Cells were plated in 60 mm dishes, and nuclear fractions were isolated as described in the manufacturers protocol using the NE PER kit. Kinase Assays Assays were done as described previously. Fleetingly, cells were serum starved immediately, lysed in a Triton X 100 based lysis load, c Abl and Arg immunoprecipitated, cleaned with a series of stringent buffers, incubated in a kinase reaction with 32P c ATP, 1 mM cool ATP and GST Crk for 409 at room-temperature. Kinase reactions were run on SDS PAGE gels, dried, exposed to film, and rings quantitated applying a STORM phosphoimager and ImageQuant Software.