Figure 2
From: Erk2 but not Erk1 regulates crosstalk between Met and EGFR in squamous cell carcinoma cell lines
Amphiregulin is an activator of EGFR and HER2. The CM of a monocytic cell line induces the release of amphiregulin in SCC9 cells. (A) EGFR IP followed by Western blot analysis of SCC9 cells treated with 100 ng/ml HGF for 24 h and with 10 ng/ml EGF for 3 min. Immunoblots for phospho-tyrosine (=pY) and EGFR are shown. Total cell lysate blotted with tubulin was used as loading control. (B) EGFR and HER2 IP followed by Western blot analysis. SCC9 cells were stimulated for 5 min with HGF CM in the presence of 5 μg/ml amphiregulin-blocking antibody (=B-AR). The blocking antibody was added to the CM 30 min prior to stimulation. Immunoblots for pY, EGFR and HER2 are shown. (C) The CM of the monocytic cell line MAD-NT induced HGF-dependent amphiregulin release in SCC9 cells. Amphiregulin release was blocked, both when MAD-NT CM was pretreated with a HGF-blocking antibody (=B-HGF), as well as when SCC9 cells were pretreated for 30 min with 1 μm of the Met inhibitor PHA-665752 (=PHA). Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. The asterisk indicate a statistically significant decrease (p < 0.05, paired t-test). (D) Western blot analysis of SCC9 cells stimulated for 10 min with HGF, MAD-NT CM, MAD-NT CM plus a HGF-blocking antibody and of SCC9 cells pretreated with the Met inhibitor PHA-665752. Immunoblots for pSAPK/JNK, pAkt, pp38 MAPK and pErk1/2 are shown. Tubulin served as loading control.