Figure 5
Analysis of signaling pathways involved in rLsa21-mediated cytokine production in mouse macrophages.
(A) RAW264.7 cells were pretreated for 30 min at 37 °C with anti-TLR2 (30 μg/ml), anti-TLR4 (30 μg/ml), or an isotype control Ab (30 μg/ml) before stimulation with rLsa21 (5 μg/ml) for 30 min. Levels of phosphorylated p38, JNK, and ERK1/2 induced by rLsa21 and degraded IκBα were analyzed by western blot using anti–phospho-p38 (p-p38), or anti–phospho-ERK1/2 (p-ERK1/2), anti–phospho-JNK (p-JNK) and anti-mouse IκBα as well as a specific control Ab for each of the unphosphorylated kinases. Data are representative of those obtained in three independent experiments. (B) TLR2−/−, TLR4−/− or TLR2−/−/TLR4−/− DKO macrophages cell lines were stimulated with rLsa21 (5 μg/ml) for 30 min. Levels of phosphorylated p38, JNK, and ERK1/2 induced by rLsa21 were analyzed by western blot as described in materials and methods. (C) RAW 264.7 cells were pretreated for 30 min with NF-kB inhibitor (SN50; 20 μM), JNK inhibitor (SP600125; 40 μM) or p38MAPK inhibitor (SB203580; 30 μM) and then stimulated with rLsa21 (2 μg/ml). After incubation, supernatants were harvested and levels of IL-6 and TNF-α were measured by ELISA. Data are representative of three different experiments. (*Indicates P < 0.05).
