Figure 4

Regulation of BTG2-enhanced IκBα degradation via activation of PI3K-Akt1. Based on our previous report [37], upstream signals of the IκBα degradation by BTG2 were investigated. (A) Immunoblot analysis showing the significant phosphorylation of Akt at Ser⁴⁷³ residue in the BTG2 expresser. To explore whether there is a crosstalk between PI3K and NFkB pathways, HeLa cells transfected with BTG2 were treated with PI3K inhibitors, LY294002 (B) and Wortmanin (C), respectively, and then regulation of IκBα degradation was examined by immunoblot analyses. Note significant inhibition of BTG2-mediated IκBα degradation after treatment with PI3K inhibitors, suggesting the cross-talk between PI3K-Akt and NFκB pathways by the expression of BTG2. (D) RT-PCR revealing the specific knockdown of Akt1, not Akt2, by the short interfering RNAs to Akt1. (E) BTG2-mediated IκBα degradation was significantly inhibited by transfection of siAkt1, whereas transfection of siControl failed to change IκBα degradation at all, supporting the specific effect of Akt1 on the activation of BTG2-mediated NFκB pathway. (F) To further specify the effect of Akt1 on the BTG2-mediated IκBα degradation, transfection of siAkt1 RNA was combined with Ad-BTG2 infection in HeLa cells, and then ChIP analysis was performed with anti-p65 antibody. As expected, interaction of p65 with κB-RE was significantly reduced in the BTG2 and siAkt1 coexpressers (1.9 vs. LacZ and siControl) than that of the BTG2 alone expresser (3.3 vs. LacZ and siControl), indicating downregulation of NFκB activation by siAkt1 (over 40%) and the activity of Akt1 at the upstream of NFkB activation in the presence of BTG2 expression. Inhibition of p65 binding to κB-RE after transfection of siAkt1 was quantified by Image J software, and the relative densities of kB-RE found in the ChIP assay based on those of the Input (ChIP/Input) were showed below the Figure 4F. The experiment was repeated (n = 3).