Figure 2. Mig6 expression is upregulated by elevated phospho-AKT in SCC-R cells.

A) Immunoblot analysis of phospho-AKT, total AKT, and loading control β-actin in SCC-S and SCC-R cells. B) SCC-R cells were treated with AKI (AKT1/2 kinase inhibitor, at 5 or 10 µM), U0126 (MEK1/2 inhibitor, at 5 or 10 µM), or DMSO (control) for 24 hrs and subjected to immunoblot analysis with indicated antibodies. C) SCC-R cells were treated with LY294002 (PI3K inhibitor, at 5 or 10 µM), rapamycin (mTOR inhibitor, at 1 or 2 µM) or DMSO (control) for 24 hrs and subjected to immunoblot analysis with the indicated antibodies. D) SCC-R cells were transfected with either scrambled siRNA or siRNA targeting PTEN for 48 hrs and subjected to immunoblot analysis. E) SCC-R cells were treated with 0.2 or 1 µM erlotinib (T0.2, T1, respectively) for 24 hrs, or pretreated with 0.2 or 1 µM erlotinib for 30 min and then co-treated with 10 ng/ml EGF for an additional 24 hrs. Mig6 levels were then evaluated with immunoblot analysis. F) SCC-R cells were treated with 10 µM LY294002, 10 µM AKT1/2 kinase inhibitor, 1 µM rapamycin, or 10 µM U0126 for 24 hrs. Cells were then treated with 10 ng/ml EGF for 30 min to induce EGFR phosphorylation and subjected to immunoblot analysis. G) Densitometric analysis of phospho-EGFR/total EGFR. DMSO-treated samples were arbitrarily assigned a value of 1 and values of the remaining samples represent fold changes of phospho-EGFR per EGFR molecule. Note that fresh Mig6 antibody recognizes a nonspecific band above the Mig6 protein, which gradually disappears after antibody re-suing or recycling.