Figure 2.

Threonyl-tRNA synthetase (TRS) activates dendritic cells (DCs) through the NF-κB and MAPK signaling pathways. (A) IκBα, IκBβ, and GAPDH expression levels, as measured by western blotting of DCs treated with TRS in a time-dependent manner (0–60 min). (B) The nuclear translocation of NF-κB p65, as detected by confocal laser scanning microscopy (LSM 800, Carl Zeiss, Oberkochen, Germany) of DCs treated with TRS (200 nM) for 1 h and then incubated with an anti-NF-κB p65 antibody (Ab), followed by staining with an Alexa Fluor 488-conjugated Ab and 4′,6-diamidino-2-phenylindole (DAPI). (C) DC culture method described in (A). Western blotting for phosphorylated JNK, p38, and ERK or unphosphorylated JNK, p38, and ERK. (D) Levels of IκBα, IκBβ, NF-κB p65, and phosphorylated NF-κB p65, as detected via western blot analysis of DCs pretreated with the MAPK inhibitors (1, 10 μM) and treated for 30 min with TRS. (E) IL-12p40 levels in the supernatant, as detected via ELISA of DCs pretreated with the indicated inhibitors (0.1, 1, 10 μM) for 30 min and treated with TRS for 20 h. The media-treated DCs without inhibitors was analyzed as a control. Experiments were conducted three times independently and are represented as the mean ± SEM of results performed in triplicate (n = 3). Statistical significance was assessed using unpaired Student’s t-test; ^###P < 0.001 compared with media-treated DCs, *P < 0.05, **P < 0.01 and ***P < 0.001 as determined by one-way analysis of variance with a Bonferroni post-test for multiple comparisons.