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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Nat Immunol. 2015 Jun 29;16(8):838–849. doi: 10.1038/ni.3205

Figure 2. IFN-γ inhibits TLR2-induced activation of MAPK-MNK-eIF4E axis.

Figure 2

(a) Immunoblot analysis of phosphorylated (p-) eIF4E and p-MNK1 in control or IFN-γ-primed macrophages treated with Pam3CSK4 (10 ng/ml) for 0-60 min; p38α serves as loading control. (b) Polysome shift analysis of NFKBIA mRNA. (c) Immunoblot analysis of HES1 in human primary macrophages pretreated for 30 min with the vehicle control dimethyl sulfoxide (DMSO) or increasing concentrations of MNK inhibitor CGP57380, then stimulated for 0, 2 or 4h with Pam3CSK4 (10 ng/ml); p38α serves as loading control. (d) qPCR analysis of HES1 mRNA in human primary macrophages (error bars, s.d.). UT=untreated; Pam= Pam3CSK4. Data are shown as means + SD of triplicate determinants and are normalized relative to GAPDH mRNA. (e) Immunoblot analysis of HES1 and phosphorylated (p-) eIF4E in human primary macrophages transfected with scrambled control small interfering RNA (siRNA) or siRNA specific for both MNK1 and MNK2 for 72h, and then stimulated for 0-4 h with Pam3CSK4 (10 ng/ml); p38α serves as loading control. (f) Immunoblot analysis of phosphorylated (p-) p38 and p-ERK in control or IFN-γ-primed macrophages treated with Pam3CSK4 (10 ng/ml) for 0-60 min; p38α and ERK serve as loading controls. (g) qPCR analysis of DUSP1, DUSP2, DUSP4, DUSP8 and DUSP16 mRNA in control or IFN-γ-primed macrophages treated with or without Pam3CSK4 for 4h (error bars, s.d.). Data are shown as means + SD of triplicate determinants and are normalized relative to GAPDH mRNA. Data are representative of at least three independent experiments (a-g).