Mass spectrometry results were analyzed with Mascot software. Western blot data images were analyzed using ImageJ with gel tool. Numerical data were processed with the Mann Whitney test. Flow cytometry was run in Cell Quest software, and data were analyzed with the Summit 4. 3 program. Ex tracted numerical data were this website analyzed statistically with the Mann Whitney test. IHC and culture data were also analyzed using the Mann Whitney test. KNK437 Ic50 of BFU E inhibition assays were calculated using the GraphPathPrism 5. 04. CBAs files were processed with FCAP Array. and data analyzed with the Mann Whitney test. Statistical significance was considered when the P value was 0. 05.
Results Inhibitors,Modulators,Libraries Identification of differentially expressed proteins using Two dimensional difference gel electrophoresis and mass Inhibitors,Modulators,Libraries spectrometry DIGE and MS were used to identify differences in the whole cytosolic proteome between PV and ET groups. Figure 2A, show three representative spots from the prote omic analyses of samples from ET and PV patients. We found 112 spots representing proteins with differential ex pression between both diseases. Identification of the spots Inhibitors,Modulators,Libraries yielded 65 proteins. Three proteins were especially inter esting in the context of our model and selected for further studies by doing a literature search on their biological Inhibitors,Modulators,Libraries function. These three differentially expressed proteins in cludes LTA4H, SERPINB1 and HSP70. Of note, HSP70 is a chaperone related to GATA 1 and eryth roid differentiation.
Most of the other spots corresponded to a large group of proteins implicated in metabolic and biochemical processes, for example, glycogen phosphoryl ase, pyruvate kinase, and lactotransferrin. Healthy donors also showed differences when compared with PV samples. There were 174 spots and 19 proteins identified. Samples from controls and ET showed differ ences in 97 Inhibitors,Modulators,Libraries spots, and six proteins were identified. Most of the proteins identified were implicated in metabolic and biochemical pathways, similarly to those observed when ET and PV were compared. A full list of the differ entially expressed proteins is summarized in Additional file 1 Table S1, Additional file 2 Table S2 and Additional file 3 Table S3. Validation of proteomic analysis by immunohistochemistry in bone marrow Bone marrow IHC analysis of HSP70, SERPINB1, and LTA4H was performed to confirm and identify the ex pression pattern found ATPase by 2D DIGE MS. Twelve bone marrow biopsies from PV patients, 10 ET JAK2 V617F positive, 13 ET JAK2 V617F negative, and 11 controls were studied. HSP70 was markedly over expressed in PV bone marrow vs. ET. A full list of the HSP70 expression sample per sample is summarized in Additional file 4 Table S4. SERPINB1 was expressed in the nucleus and cyto plasm of the granulocytes.