Ag1478

AG1478 was purchased from Wako (Osaka, Japan), gefitinib was obtained from AstraZeneca, erlotinib was from ChemieTek (Indianapolis, USA), and KT5720 and LY294002 were from Sigma-Aldrich. The chemical library included 30,000 low-molecular weight compounds supplied by the ChemBridge Screening Libraries (San Diego, CA, USA). Rabbit anti-human EGFR (D38B1), rabbit anti-phospho-EGFR Tyr1068 (D7A5), mouse anti-ubiquitin (P4D1), and HIF-1α monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, USA). Mouse anti-β-actin (AC-15) monoclonal antibody was obtained fom Abcam (Cambridge, UK). Peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were supplied by Jackson Immunoresearch (West Grove, PA, USA).

The dorsal skin of mice was shaved and 100 μg DMBA or vehicle (200 μL acetone) was applied twice a week. Skin tissues were collected 2 weeks after the initiation of the DMBA treatment. One hundred micrograms of the EGF receptor antagonist AG1478 (Wako) was applied to the dorsal skin of mice 3 hours before sampling. The amount of the active form of Ras was measured using an ELISA Kit (STA-440; Cell Biolabs, Inc.). The values of absorbance at 450 nm were normalized by total protein concentrations in the samples. Protein concentrations were measured using the BCA assay (Thermo Fisher Scientific).