CHIR-258 was determined by Bio-Rad reagent measured using BSA as standard

0 mM NaCl, 1 mM EDTA, and the protease inhibitors 1 mM PMSF, 2 g / ml aprotinin, CHIR-258 2 g / ml leupeptin, 50 mM NaF, 1 mM sodium venadate and 500 g / ml benzamidine. The cells were incubated with lysis buffer usually for 30 minutes on ice with gentle stirring vortex. The cell lysate was centrifuged at 4 for 10 minutes. The supernatant was removed, and in a separate R Hrchen, and the protein concentration was determined by Bio-Rad reagent measured using BSA as standard. Tests Abl kinase JAK2 and Bcr. JAK2 kinase assay was by the methods of Xie et al.9 and Sandberg et al.44 32Dp210 without cell lysate by treating cells with lysis buffer containing 20 mM Tris-HCl, 100 mM NaCl is carried out, 1% NP40, and protease inhibitors .
Aliquot of each lysate was detergent Eppendorfgef with 500 g of L lysis buffer and extracted protein/500 screened with protein G-agarose conjugate. The supernatant was mixed with 50 l Abl antique Body incubated for 1 hour and 30 L protein G-agarose beads for 1 hour for the Immunpr Zipitation cooperation Bcr Abl/Jak2. Followed by washing with lysis buffer by washing with CHIR-124 kinase buffer the agarose were suspended in kinase buffer. Added varying amounts ON044580 and incubated for 10 minutes, and the reaction was started by addition of 2.5 mM ATP. The reaction was continued for 30 minutes at room temperature, and the reaction was stopped by addition of 2 x sample buffer. The kinase reaction signals were analyzed by Western blot with an antique Rpern pJak2 Tyr1007/1008 detected. Bcr Abl kinase autophosphorylation was determined by the method of Bcr Abl Bartholomeusz al.
63 and by Immunpr zipitation With antique Rpern P6D performed. The Immunpr Zipitate were incubated with different amounts of ON044580. Kinase reactions were performed with more cold ATP, Mg, and 1 mM dithiothreitol at 30 w Introduced during 30 minutes. Kinase activity T was determined by Western blotting with an antique Detects body against pTyr. In vitro kinase assay with Abl kinase JAK2 and recombinant proteins. Recombinant Jak2 kinase Abl kinase, and were tested in vitro with the methods ver Changed. Recombinant Jak2 kinase assay: Recombinant Jak2 was preincubated for 10 minutes with different amounts of ON044580 in an incubation mixture as described above for the kinase assay cold.
After 10 minutes, the reaction through the cold ATP, 10 Ci / test 32P gamma ATP and 5 M peptide substrate described Jak2 initiated originally 9 and the incubation was continued for 10 minutes at 30. The reaction tubes were kept on ice, 250 g of BSA was added, and then an equal volume of trichloroacetic Acid and incubated for 30 minutes. After centrifugation, the pellet was washed twice with 20% TCA, and the pellet was resuspended to Z Select gamma 32P ATP in the pellet in a gamma-Z Incorporated probes used. Recombinant Abl kinase assay: To determine the Abl kinase, 20 ng recombinant kinase Abl was same with kinase buffer as for the determination of JAK2 kinase used mixed. A different H eh The ON044580 was added to the incubation mixture and incubated for 10 minutes. The reaction is started by adding the substrate of the Abl kinase, 5 M unlabeled ATP and radiolabeled ATP. The reaction was stopped by adding 5 L of 3% phosphoric acid From the mixture is stopped, and 10 L of the mixture was dropped onto Whatman fi

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