Fig. 4: TRAF6 regulates the EGF-induced signaling pathway through EGFR.

a, b, c Silencing of TRAF6 attenuates p-EGFR and EGFR expression. HaCaT cells expressing sh-TRAF6#1,2 were starved for 36 h and then treated with EGF (100 ng/ml) for 30 min, and a protein array was performed to assess the effect of TRAF6 on the EGFR signaling pathway (a) or cells expressing sh-TRAF6 were starved for 36 h and then treated with EGF (100 ng/ml) for various times, as indicated. Immunoblotting was used to detect protein expression with the indicated antibodies. Anti-β-Actin or Gad were used to verify equal loading of protein (b, c). The histograms indicated relative MMP-2 and MMP-9 expression, as means ± S.D. Significant differences were evaluated using a one-way ANOVA and the asterisk (*) indicates a significant difference (p < 0.05) (lower panel). d TRAF6 regulates AP-1 activity. Flag-TRAF6-WT and Flag-TRAF6-DN cells were transfected with the AP-1-luciferase reporter gene (100 ng), as well as the Renilla luciferase gene (20 ng) for normalization. At 30 h after transfection, the firefly luciferase activity was determined in cell lysates and normalized to the Renilla luciferase activity. Significant differences were evaluated using Student’s t-test, and the respective asterisks indicate a significant difference (p < 0.05) (upper panel). TRAF6-silenced cells were co-transfected with a plasmid mixture containing the AP-1 luciferase reporter gene (0.8 µg) and the Renilla luciferase gene (0.2 µg) for normalization. At 20 h after transfection, cells were starved for 16 h and then treated with EGF (20 ng/mL) for various times, as indicated. Firefly luciferase activity was determined in cell lysates and normalized against Renilla luciferase activity. Significant differences were evaluated using Student’s t-test, and the respective asterisks indicate a significant difference (p < 0.05) (lower panel)