Figure 6. TPL-2 catalytic activity is required for TNF activation of MKK3/6, p38α and MK2 in macrophages.

(A) WT and Map3k8^D270A/D270A macrophages stimulated with TNF ± PD0325901 for 15 min. Total cell lysates were immunoblotted for the indicated antigens. (B) WT and Nfkb1^SSAA/SSAA macrophages were stimulated with TNF for 15 min. Total cell lysates were immunoblotted for the indicated antigens. (C) WT and Map3k8^D270A/D270A macrophages were stimulated separately with a panel of agonists. Activation loop phosphorylation of p38α was quantified by infrared scanning of immunoblots. Data, normalized to total p38α levels, are plotted (n = 5; SEM). P-values were calculated by two-way ANOVA. **P ≤ 0.005. MKK6 phosphorylation was determined by immunoblotting of total cell lysates (lower panels). (D) WT and Map3k8^D270A/D270A macrophages stimulated with TNF for 15 min. Total cell lysates were immunoblotted for the indicated antigens. (E) WT and Map3k8^D270A/D270A macrophages stimulated with LPS for 15 min. Total cell lysates were immunoblotted for the indicated antigens. Immunoblots in panels A, C, D and E are representative of at least three independent experiments, and in panel B representative of two independent experiments.