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. 2012 Jan 9;7(1):e29993. doi: 10.1371/journal.pone.0029993

Figure 4. Reduced NFκB activation by TNFα in primary hepatocytes from Sharpin-deficient mice.

Figure 4

A. NFκB activity assay. Hepatocytes isolated from wt and Sharpin-deficient mice were transfected with reporter vectors encoding CMV-driven Renilla luciferase and NFκB promoter dependent Photinus luciferase. After incubation with TNFα for 24 hours, cell lysates were analyzed for activity of both luciferases; NFκB promoter activity is depicted as the ratio between Photinus and Renilla luciferase activities from three independent experiments. ANOVA/Tukey test **,*** p<0.01/0.001, respectively; n = 3. B. Real time RT-PCR analysis. Hepatocytes isolated from wt and Sharpin-deficient mice were incubated with TNFα for 24 hours. After isolation of RNA, levels of the mRNA coding for A20 were determined by quantitative real time RT-PCR relative to levels of mRNA coding for ATP synthase beta. C,D. Phosphorylation of IκBα. Hepatocytes were incubated with TNFα for the times indicated. Cell lysates were analyzed by Western blotting using the antibodies indicated (C). In D, the amount of phospho-IκBα from three independent experiments as shown in C is quantified. The intensity of phospho-IκBα signals was normalized on GAPDH signals; in a second step, data were normalized on the maximum value (in each case the 10 minute time point in wt cells). *, +/+ data significantly different from −/− data; p<0.05; n = 3; t-test. E,F. Degradation of IκBα. Hepatocytes were incubated as described in C, and analyzed by Western blotting with the antibodies indicated. In F, the amount of the amount of IκBα from three independent experiments as shown in E is quantified. In this case the amount of IκBα in the control experiment (no TNFα incubation) was set as 100% for each genotype. G. Nuclear translocation of NFκB. Hepatocytes were incubated with TNFα as indicated; cytosolic and nuclear fractions were prepared and analyzed by Western blotting with antibodies against GAPDH (cytosolic marker), Lamin B (nuclear marker) and p65/RelA. Note that the intensity of the signal for the p65/RelA component of NFκB increases in nuclei of TNFα-incubated wt, but not Sharpin-deficient cells. WT mice (+/+); Sharpin-deficient mice (−/−).