Figure 4. Effects of adenoviral-mediated overexpression of wild-type IκBα, IκBα S32,36A and β-galactosidase on the regulation of the NFκB pathway and G6Pase gene expression.

H4IIE cells were infected with virus expressing wild-type IκBα (Ad-IκBα wt), IκBα S32,36A (Ad-IκBα S32,36A) or β-galactosidase (Ad-β-Gal) and incubated with TNFα, insulin, Bt₂cAMP (cAMP) and dexamethasone (Dex), as indicated, prior to the isolation of cell lysates, nuclear extracts and RNA. (A) Western blot of cell lysates using anti-IκBα antibodies. (B) ELISA of activated NFκB in nuclear extracts isolated from H4IIE cells treated with or without TNFα. Data are expressed relative to the absorbance with Ad-IκBα wt in the absence of TNFα, which was set as 1; *P<0.05 compared with relative activation in the absence of TNFα. (C) Northern blot. RNA (15 μg) isolated from cells infected and incubated as indicated was electrophoresed, transferred on to nylon membranes and hybridized using a random-primed ³²P-labelled probe for G6Pase. Actin was used as a normalization control. (D) Densitometric analysis of three Northern blots (means±S.E.M.; n=3); *P<0.05 compared with expression in the absence of insulin or TNFα. The level of expression of Ad-β-Gal-infected cells in the presence of dexamethasone and Bt₂cAMP was set as 100.