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. 2000 May 9;97(10):5243–5248. doi: 10.1073/pnas.97.10.5243

Figure 4.

Figure 4

Dependence on IKKγ and expression of MAP3Ks and GCK in wild-type (+/+) and mutant (−/−) ES cells. (A) Wild-type and Ikkγ- (only a single Ikkγ allele was disrupted, but as Ikkγ is an x-linked gene and the ES cells we used were XO, this resulted in complete loss of IKKγ expression) ES cells were left untreated (0 time point) or treated with TNFα or IL-1 as described above. At the indicated times, cell lysates were prepared and IKK activity was determined by using an IKKα monoclonal antibody and immunocomplex kinase assay. (B) Exponentially growing ES cells or 3T3 fibroblasts were lysed, and equal amounts (50 μg) of total cell lysates were resolved by SDS/PAGE, transferred to a nitrocellulose membrane, and probed with antibodies to ASK1, TAK1, MEKK2, or GCK (Santa Cruz Biotechnology). All of these antibodies were found to specifically recognize their cognate antigens based on immunoblot analysis of cells transiently transfected with expression vectors for the different kinases (not shown).