Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury
J. Clin. Invest. Tom Luedde, et al. 115:849 doi:10.1172/JCI23493 [Go to this article.]

Figure 3
NF-κB activation in the liver upon TNF-α stimulation is blocked in mice lacking Nemo but not Ikk2. (A) Liver nuclear protein extracts (5 μg) from the indicated mice and time points after TNF-α stimulation were subjected to a gel-retardation assay with an NF-κB consensus probe. In lanes 10/22/34 and 11/23/35, antibodies for the NF-κB subunits p50 or p65 were added as indicated as supershift control. The figure depicts results from 3 different assays. (B) I-κBα degradation in the different mouse groups was assessed by Western blot analysis with 50 μg of whole cell liver protein extracts before and 10 minutes after TNF-α stimulation using an antibody against I-κBα or α-tubulin (as loading control). (C) I-κBα phosphorylation was detemined after TNF-α stimulation by subjecting 50 μg of proteins to a Western blot analysis with an antibody detecting I-κBα phosphorylated at Ser32. (D) Evaluation of IKK activity. Proteins (300 μg) from mice stimulated with TNF-α were IP with a Nemo-antibody and subjected to a kinase assay using a truncated glutathione-S-transferase–I-κBα(1–54) protein as substrate. (E) Gel-retardation assay with an NF-κB consensus site using 5 μg of nuclear protein extracts from primary hepatocyte cultures. Results are representative of those obtained in mice (n = 4). (F) JNK activity was measured by Western blot using protein from mice stimulated with TNF, LPS, and ConA as indicated. Antibodies detecting c-Jun phosphorylated at Ser63 or JNK phosphorylated at Thr183/Tyr185 as well as nonphosphorylated JNK1 and α-tubulin as loading control were used.