TR FRET emission ratios were determined on a BMG Pherastar fluorescence plate reader using these parameters: excitation at 340 nm, emission 520 nm and 490 nm, 100 us lag time, 200 us integration map kinase inhibitor time, emission proportion Em 490. All data were analyzed and plotted using Graphpad Prism 4. High-throughput Microscopy Cells were plated at 7500 cells/well in 96 well microscopy plates in proposed media for 24 hours, and then deprived in media missing serum for 16 hours. Cells were pre treated for 180 minutes with 10 fold stock solutions of JNK inhibitors and for 10 min with get a grip on materials MK2206, PD0325901, SB239063, KIN001 040 and KIN001 208 and treated with 10 fold stock solutions of IGF 1, IL 6, TNF or anisomycin for 60 minutes. Cells were fixed last year paraformaldehyde for 10 min at room temperature and washed with PBS T. Cells were permeabilized in methanol Gene expression for 10 min at room temperature, washed with PBS T, and plugged in Odyssey Blocking Buffer for 1 hour at room temperature. Cells were incubated overnight at 4 C with antibody specific for pP38, Akt, cJUN, Erk1/2 and pSTAT3, pRSK1 and pMSK1 and NF W diluted 1:400 in Odyssey Blocking Buffer. Cells were washed three times in PBS T and incubated with rabbit specific secondary antibody labeled with Alexa Fluor 647 diluted 1:2000 in Odyssey Blocking Buffer. Cells were washed once in PBS T, once in PBS and incubated in 1:1000 Whole Cell Stain option and 250 ng/ml Hoechst 33342. Cells were washed 2 times with PBS and imaged in an imageWoRx high throughput microscope. Information was plotted using DataPflex. Binding Kinetics assay A375 cells were pre-treated with 1uM compound for the indicated amounts of time. Remove the medium Aurora B inhibitor and wash 3 times with PBS. Re-suspend the cell pellet with 1 mL Lysis Buffer. Move end to end for 30 min at 4 C. Lysates were removed by centrifugation at 14000 rpm for 15 min in the Eppendorf. The cleaned lysates serum blocked into Kinase Buffer using Bio Rad 10DG colums. The total protein concentration of the solution filtered lysate must be around 5 15 mg/ml. Cell lysate was described with the probe from ActivX at 5 uM for 1-hour. Samples were paid down with DTT, and cysteines were blocked with iodoacetamide and gel filtered to change the buffer and remove excess reagents. Antibodies Rabbit polyclonal antibodies against total pan JNK isoforms, phospho pan JNK isoforms,, total p38 or phospho p38 MAPK,, total c Jun, phospho c Jun, and phospho MSK1 were from Cell Signalling technology. Main antibodies were used at a concentration of 1 ug/ ml, diluted in 50-peso skimmed milk in TBST and incubated over night at 4 C. Detection of immune complexes was performed using a sophisticated chemiluminescence reagent and horseradish peroxidase conjugated secondary antibodies.