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. 2019 May 10;9:158. doi: 10.3389/fcimb.2019.00158

Figure 4.

Figure 4

EspC induced the activation of MAPK and NF-κB. (A) RAW264.7 cells were incubated with EspC (5 μg/mL) for the indicated times (0, 5, 10, 15, 30, 45, 60, 75, 90, and 120 min). Cell lysates were then prepared, and the phosphorylation and activation of p38 MAPK, JNK, and ERK1/2 were examined by western blotting using specific antibodies targeting phospho-p38 MAPK (p-p38 MAPK), p38 MAPK, phospho-JNK (p-JNK), JNK, phospho-ERK1/2 (p-ERK1/2), and ERK1/2. (B) After incubation with EspC (5 μg/mL) for the indicated times, RAW264.7 cells were subjected to a protocol to separate cytoplasmic and nuclear fractions. The effects of EspC on the translocation of NF-κB from the cytoplasm to the nucleus and total IκBα levels in cytoplasmic extracts were determined by western blot analysis. (C) RAW264.7 cells were pretreated with blocking antibodies before stimulation with EspC (5 μg/mL). Western blotting analysis was performed to analyze the levels of p-p38 MAPK, p-JNK, p-ERK1/2, p38 MAPK, JNK, and ERK1/2. The relative band intensity of each protein was analyzed by Image J. Data are representative of three independent experiments and are shown as means ± SEMs (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.