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. 2016 May 6;7(24):36865–36884. doi: 10.18632/oncotarget.9211

Figure 5. ERK phosphorylation is required for the JWA/PEA3 signaling axis.

Figure 5

A, B. NCI-N87 cells were transfected with FLAG-JWA or vector for 36 h and then treated with U0126 (25 μg/ml) or DMSO for 6 h and EGF (100 ng/ml) for 20 min. HER2 luciferase reporter promoter plasmids (pNeuLite) were co-transfected with vector or FLAG-JWA into NCI-N87 cells. The luciferase activity was normalized to pRL-CMV. The HER2 promoter luciferase activity in vector-treated cells was used as a control to calculate the relative HER2 luciferase activity (A). The PEA3, p-ERK, JWA, and HER2 protein levels were examined by western blot analysis (B). C. The MEK (U0126; 25 μM) and/or PI3K (LY294002; 50 μM) inhibitors or control DMSO were added to the cells for 6 h, and the cells were then transfected with FLAG-JWA or vector for 36 h. The cells were then harvested to detect HER2, p-AKT, p-ERK, p-PAK1, FAK, COX2, or FLAG by immunoblotting. The data in the bar graphs represent the mean and SD of three independent experiments. * P<0.05 and ** P<0.01; Student's t-test. Arrows indicate the interested band.