We then chosen the following proteins Hsp, Hsp, Peroxiredoxin , Annexin A , Fuse binding protein , Rho GDP dissociation inhibitor , Carbonic anhydrase II and Malic enzyme . As shown in Fig. A, Hsp, Hsp, Prdx , Anxa and Fubp protein expression decreased in KCLR cells, whereas Arhgdia, Ca and Me protein expression improved in KCLR cells. We examined the results of three Western blot experiments by densitometry implementing Gapdh protein expression to normalize the data , consequently validating DIGE analysis. Considering our analysis showed down regulation of Hsp and Hsp in KCLR cells we measured the expression of other members within the heat shock protein family members, namely Grp and Hsp, which are differentially expressed in cancer cells, which includes leukemia, and are resistant to apoptosis . The expression of these two proteins, measured by Western blot evaluation, was diminished in KCLR cells . The down regulation of Hsp, Hsp and Anxa proteins in KCLR cells could occur at genetic level as demonstrated by a preliminary research on the gene expression profiles of imatinib resistant and imatinib delicate KCL cells .
On top of that, quantitative RT PCR showed a significant reduce in the expression from the Annexin A gene in KCLR cells . Mainly because Hsp expression is beneath the handle from the Hsf transcription activation factor, we measured the expression of Hsf by Western blot analysis. The expression of Hsf was reduced in KCLR as confirmed by densitometric Nutlin-3 molecular weight analysis . These information propose that down regulation of Hsp may be mediated by an Hsf dependent mechanism. Employing gene expression profile examination we also noticed that SHP expression was reduced in KCLR . Therefore, we measured the levels of Shp protein in KCLR and KCLS cells. Western blot examination showed that Shpwas down regulated in KCLR cells . Considering that Shp could act being a damaging regulator of cell proliferation currently being essentially an antagonist of Shp , we investigated the expression degree of Shp in KCLR and KCLS cells.Western blot evaluation showed that the degree of Shp was comparable in resistant and sensitive cells .
Simply because a number of proteins which have been differentially expressed in KCLR and KCLS cells are concerned inside the modulation of cellular proliferation and apoptosis,we investigated the degree of activation of Erk ?. To this aim, we measured the degree Motesanib kinase inhibitor of Erk and its phosphorylated type by Western blot examination. As shown in Fig. C and D, the degree of total Erk? was comparable in KCLR and KCLS cells. In contrast, the level of phosphorylated Erk?was higher in KCLR cells than in KCLS cells , which suggests that Erk was constantly activated in KCLR cells. Lately, a chemical proteomic screen for imatinib interactors uncovered a non kinase target, the oxidoreductase Nqo. We for that reason analyzed the expression of Nqo in KCLR and KCLS cells.