| HSP70 and HSP90 play very important roles in cell signal transduction pathways. The studies show that the protein interaction between HSP90 / HSP70 and PKB has critical impact on adjusting the activity of PKB during cell stress. But the molecular mechanism of the interaction between HSP70 / HSP90 and PKB is still unclear.Firstly, we treat HEK293T cells with heat shock stimulation and detect the interaction between HSP70 / HSP90 and PKB in HEK293T cells by using of PULL-DOWN. The results show that the association of PKB and HSP70 increased significantly after Geldanamycin inhibits the association of HSP90 and PKB in the cells. From this result, we speculate that there maybe have a certain compensation effect to increase the combination between HSP70 and PKB enen if inhibiting the combination of HSP90 and PKB during cell stress. However, whether is there a compensation for combining between HSP90 and PKB after inhibiting the combination between HSP70 and PKB? This deserves our further study.Due to the poverty of ideal HSP70 specific inhibitor, the HSP70 protein-binding domain is truncated to construct two kinds of HSP70 mutants. One mutant only contains ATP domain of HSP70, the other contains ATP domain and CT domain of HSP70. After the identification by two restriction enzymes digestion and DNA sequencing, both of two eukaryotic expression recombinants are to be successfully constructed, which named truncated-ATP and truncated-ATP-CT. We transfect these recombinants into HEK293T cells. There are four groups: (1) control group (nontransfection group); (2) HSP70 total length plasmid transfection group; (3) truncated-ATP plasmid transfection group; (4) truncated-ATP-CT plasmid transfection group. WESTERN-BLOT detect HSP70 expression, the results show that both two recombinants are successfully expressed in HEK293T cells. These results will lay the foundation for elucidating the mechanism of the interactions between HSP70 /HSP90 and PKB . |
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