Figure 4. Kras upregulates p22^phox expression via the Tak1-NF-κB pathway.

(a) The expression of p-Tak1, Tak1, p-NF-κB/p65 and NF-κB/p65 was analysed by immunoblotting in HPNE, HPNE/Kras^G12V and HPNE/Kras^G12V/shp16 cells. (b) The expression of Tak1, p-NF-κB/p65, NF-κB/p65 and p22^phox was analysed by immunoblotting in AsPc-1/i-Tak1shRNA cells with Dox induction. (c) The expression of Kras, p-NF-κB/p65, NF-κB/p65 and p22^phox was analysed by immunoblotting in mPDAC/iKras cells with Dox induction. (d) The NF-κB/p65 and p22^phox mRNA expression was analysed by qPCR in HPNE/Kras^G12V/shp16 cells transfected with NF-κB/p65 siRNAs. (e) The expression of phosphorylation-defective mutant IκBα, p-NF-κB/p65, NF-κB/p65 and p22^phox was analysed by immunoblotting in wild-type (WT) and IκBα-mutant (Mu) AsPc-1 and Panc-28 cells. (f) NOX activity was compared in IκBα wild-type and mutant AsPc-1 and Panc-28 cells. (g) The sequences of human p22^phox promoter regions are presented with NF-κB binding sites (BS-1 and BS-2) indicated. (h) The activities of NF-κB binding sites BS-1 and BS-2 in p22^phox promoter were analysed in indicated cells by ChIP and qPCR assay. IgG was used as a negative control. (i) The activities of NF-κB binding sites BS-1 in p22^phox promoter were analysed in indicated cells by ChIP assay and qPCR. (j) The expression of p-NF-κB/p65, NF-κB/p65 and p22^−phox was analysed by immunoblotting in AsPc-1 cells treated with TNF-α (10 ng ml⁻¹) for 48 h. β-actin was used as the internal loading control. Data in d,f,h,i are presented as mean±s.d. (n=3). **P<0.01 for indicated comparison (Student unpaired t-test).