Figure 3

Genetic knockdown of GSK-3 by siRNA results in disruption of NF-κB activity. A. Western blot analysis of expression of NF-κB target genes XIAP, BcL-X_L, and cyclin D1 in PANC-1 cells after transient knockdown of GSK-3 isoforms; α(10 nM siRNA), β (80 nM siRNA) or both. Expression level of total GSK-3 α or β isoforms confirms the genetic knockdown of the specified gene. Increased cytosolic β-catenin expression confirms GSK-3 inhibition. The change in the expression level of the proteins is compared against untreated or scrambled siRNA (negative control) treated controls. α-tubulin is used as loading control. B. Effect of genetic disruption of GSK-3 on basal and TNF-α induced NF-κB activity measured by luciferase reporter assay. PANC-1 cells were genetically knocked down for GSK-3 isforms α, β or both, and subsequently were co-transfected with TA-LUC NF-κB and β-gal (internal control) constructs. The cells were then exposed to TNF-α(30 ng/mL, 4 h). The normalized values are relative to the untreated control which is represented by dotted line (indicating basal level of NF-κB activity). Scrambled siRNA with or without TNF-α treatment is used as control. Each column represents mean for at least four experiments, each with three replicates; error bars = ± SEM. (*) significant: (p < 0.0005) when compared to untreated control. (**) significant: (p < 0.0001) when compared to TNF-α treatment. Western blot analysis of expression of GSK-3α and β isoforms in the above cells confirms successful knock down of the target genes.