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. Author manuscript; available in PMC: 2015 Jun 19.
Published in final edited form as: Cancer Res. 2008 Oct 1;68(19):8004–8013. doi: 10.1158/0008-5472.CAN-08-0280

Figure 4. Inhibition of EGFR by AG1478 blocks MAP kinase activation.

Figure 4

Figure 4

Figure 4

A. The mitogenesis assay was performed as described in “Materials and Methods”. Quiescent FET cells were treated with increasing concentrations of AG1478 as indicated (mean ± s.d.; n=3). B. Cells were treated with various concentrations of AG1478 for 30’ prior to TI release (60’) and the lysates were incubated with the EGFR antibody. The antibody-antigen complex was then precipitated with protein A-agarose and resolved by 7.5% SDS-PAGE followed by immunoblotting with the phosphotyrosine antibody RC-20. EGFR activation was also detected by using an anti-phospho-EGFR Y1173 antibody (Santa Cruz) (B). C. Lysates were also analyzed for the activation of Erk, p38 and Akt. IGF1R phosphorylation was measured either by an anti-phospho-IGF1R antibody (shown) or by incubating cell lysates with PY-20 antibody and the immunocomplex was then precipitated with protein A-agarose and resolved by 7.5% SDS-PAGE followed by IGF1R immunoblotting (not shown). D. Inhibition of IGF1R activation down-regulated phospho-Akt: Quiescent cells were treated with 1 μM of PQIP for 30’ prior to TI release (60’) and the lysates obtained were analyzed for the activation of IGF1R and Akt. Total Akt was also used as a loading control. Q: quiescence. TI: supplemental McCoy’s medium containing transferrin and insulin. FET cells were transfected with IGF1R siRNA to down-regulate IGF1R as described in “Materials and Methods”. Lysates were analyzed for IGF1R and the activation of IGF1R and Akt. Actin was used as a loading control. PQIP does not block EGFR phosphorylation in EGF stimulated cells: cells were treated with 1 μM of PQIP for 30’ prior to TIE release (60’) and the lysates obtained were analyzed for the activation of IGF1R and EGFR. Actin was used as a loading control. N: no change. TIE: supplemental McCoy’s medium containing transferrin, insulin and EGF. Tarceva fails to affect IGF1R signaling: cells were treated with 1 μM of Tarceva for 30’ and the lysates obtained were analyzed for the activation of EGFR and IGF1R.