Click on image to view larger version.
Figure 3. Activation of ERK by synthetic lipid A is dependent on Cot/Tpl2. (A) Osteoblasts isolated from calvaria of the wild-type and cot/tpl2-/- mice were treated with 1 µg/mL synthetic lipid A for the indicated times. ERK phosphorylation was measured by Western blot with an mAb specific for the phosphorylated form of ERK. The same filters were re-blotted with an anti-ERK antibody to show consistent amounts of the kinase. (B) Osteoblasts isolated from the wild-type and cot/pl2-/- mice were treated with 1 µg/mL synthetic lipid A for the indicated times. Synthetic lipid-A-mediated ERK kinase activation was measured by the in vitro kinase assay with myelin basic protein (MBP) as a substrate. (C) Osteoblasts isolated from the wild-type and cot/tpl2-/- mice were treated with 1 µg/mL synthetic lipid A for the indicated times. Phosphorylation of p38 and JNK was measured by Western blot with the use of Abs specific for the phosphorylated forms of p38 and JNK, respectively. The same filters were re-blotted with an anti-p38 or JNK1 Ab to show consistent amounts of the kinases. Slightly increased basal p38 phosphorylation in cot/tpl2+/+ mice in Fig. 3C was not consistently observed in repeated experiments.
