Fig 3.

MK2 and -3 regulate Akt phosphorylation in response to multiple TLR agonists. (A) BMDMs were cultured from wild-type or MK2/3 knockouts and stimulated with 1 μg/ml of Pam₃CSK₄, 10 μg/ml of poly(I·C), 100 ng/ml of LPS, 1 μg/ml of CL097, 2 μM CpG-ODN1826, 200 μg/ml of zymosan, 10 μg/ml of curdlan, 50 ng/ml of GM-CSF, or 10 nM C5a for 30 min. Cells were then lysed and the levels of the indicated phosphorylated and total proteins determined by immunoblotting. Gray arrowheads indicate MK2 bands, while the open arrowhead indicates a nonspecific band. Blots are representative of a minimum of two independent experiments. (B) BMDMs were cultured from wild-type mice and stimulated for 15 min with LPS or H₂O₂ as indicated. Cells were then lysed and the levels of phospho-Ser473 Akt and total ERK1/2 measured by immunoblotting on the same membrane. Akt Ser473 phosphorylation was quantified as described in Materials and Methods. Results of the quantification are shown on the left, and a representative membrane is shown on the right. Error bars represent the standard deviations of 3 independent experiments.