Figure 4.

Activation of MAPK and NF-κB signaling pathways in DCs treated with PJ-EVs. (A) DCs were incubated with PJ-EVs (30 μg/mL) for different times. After incubation, these cells were lysed for protein extraction. SDS-PAGE and immunoblotting analyses were performed to assess the maturation signal using phosphorylated antigen-specific and total antigen-specific antibodies against ERK, JNK, p38, IĸB-α, and NF-κB. Data are representative of three independent experiments. (B) Western blotting data were assessed using the Image J software to compare the phosphorylated and total forms. (C,D) DCs pretreated with MAPK and NF-κB signaling pathway inhibitors, U0126 (ERK inhibitor, 10 μM), SP600125 (JNK inhibitor, 20 μM), SB203580 (p38 inhibitor, 20 μM), and Bay11-7082 (NF-κB inhibitor, 10 μM), were incubated with PJ-EVs (30 μg/mL) for 18 h. (C) Expression levels of surface molecules (CD80, CD86, MHC-I, and MHC-II) on cells analyzed by FACS. (D) Levels of extracellular cytokines (TNF-α and IL-12p70) were analyzed using ELISA. The results are indicated as mean ± SD (n = 4 samples) of three representative experiments. ** p < 0.01, or *** p < 0.001. SD: standard deviation.