96 | Chapter 4 Figure 4. AKT is not involved in regulating RAG1 expression following DNA damage. (A) Western blot analysis of STI571-treated BV173 BCR-ABL-positive B-ALL cells treated as indicated. DNA damage-induced RAG1 down-regulation could not be prevented by pretreatment with the AKT kinase inhibitor GSK690693. Effective inhibition of AKT kinase was shown by accumulation of AKT Ser473 phosphorylation and diminished mTOR Ser2448 phosphorylation. The b-actin blot was used to control for loading. (B) Western blot analysis of STI571-treated BV173 cells treated as indicated. Pretreatment with 10 mM AKT inhibitor VIII did not prevent DNA damage-induced RAG1 downregulation. The b-actin blot was used to control for loading. (C) Realtime quantitative RT-PCR analysis of Rag1 mRNA expression in A70 WT mouse v-Abl pre-cells treated as indicated, means ± SEM are shown (n=4, * p<0.05, one-sample t test). (D) Western blot analysis of STI571-treated BV173 cells transduced with empty vector (EV), wild type AKT (AKT-WT), or kinase-dead AKT (AKT-KD), and treated as indicated. The AKT blot was used to show AKT overexpression; mTOR Ser2448 phosphorylation was diminished upon DNA damage in AKT-KD overexpressing cells. The b-actin blot was used to control for loading. Treatment with GSK690693 resulted in the accumulation of AKT Ser473 phosphorylation, probably due to disturbed negative feedback regulation impinging on AKT, as was previously reported for this particular AKT kinase inhibitor39-41. Moreover, treatment with either AKT inhibitor had no effect on the down-modulation of Rag1 mRNA in A70 mouse v-Abl pre-B cells following DNA damage (Figure 4C). Furthermore, inhibition of the PI3K p110∂ isoform, which is an upstream regulator of AKT, by the specific inhibitor CAL101
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