TR FRET emission rates were determined on a BMG Pherastar fluorescence plate reader using the next parameters: excitation at 490 nm, emission 520 nm and 340 nm, 100 us lag time, 200 us integration reversible HCV protease inhibitor time, emission rate 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 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 substances 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 in 2% paraformaldehyde for 10 min at room temperature and washed with PBS T. Cells were permeabilized in methanol locomotor system for 10 min at room temperature, washed with PBS T, and blocked 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 B 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 answer and 250 ng/ml Hoechst 33342. Cells were imaged in a imageWoRx high throughput microscope and washed two times with PBS. Data was plotted using DataPflex. Binding Kinetics analysis A375 cells were pre treated with 1uM compound for the indicated amounts of time. Take away the medium Oprozomib concentration and wash 3 times with PBS. Re-suspend the cell pellet with 1 mL Lysis Buffer. Turn end to end for 30 min at 4 C. Lysates were removed by centrifugation at 14000 rpm for 15 min within the Eppendorf. The eliminated lysates solution filtered into Kinase Buffer applying Bio Rad 10DG colums. The total protein concentration of the serum blocked lysate should be around 5 15 mg/ml. Cell lysate was labeled using the probe from ActivX at 5 uM for 1-hour. Samples were paid off with DTT, and cysteines were blocked with gel and iodoacetamide filtered to remove excess reagents and change the buffer. 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. Key antibodies were diluted in five hundred skimmed milk in TBST, employed at a concentration of 1 ug/ ml and incubated over night at 4 C. Detection of immune complexes was performed using horseradish peroxidase conjugated secondary antibodies and an enhanced chemiluminescence reagent.