Figure 1
Constitutive phosphorylation of EGFR in non-small cell lung cancer cell lines (NSCLC). (A) Constitutive phosphorylation of EGFR at Y-845 and Y-992. Western blots from lysates of unstimulated NSCLC cell lines and chronic lymphocytic leukemia (CLL) cells were probed with anti-phospho-EGFR (Y-845), anti-phospho-EGFR (Y-992), anti-EGFR or anti-actin antibodies as loading controls. (B) EGFR triggered autophosphorylation is not responsible for constitutive EGFR (Y845) or (Y-992) phosphorylation in Calu3. Calu3 cells were incubated with EGFR kinase inhibitor @ 1 μM AG1478, or an equal volume of DMSO solvent, for 1 hour with or without addition of 0.5 μg EGF in the final 10 minutes before lysates were prepared and Western blotted with anti-phospho-EGFR (Y845 and Y-992), anti-phospho-Akt (ser-473), anti-Akt, or anti-actin. (C) EGFR ligands are not responsible for constitutive phosphorylation in Calu3 cells. EGFR neutralizing antibodies, LA1 at 12.5, 25 or 50 μg were incubated for 18 hours with Calu3 cells with or without 100 ng EGF in the final 5 minutes before lysates were prepared and Western blotted. (D) Transactivation by membrane associated ligands was not responsible for constitutive phosphorylation of EGFR Y-992 or downstream phosphorylation of Akt or Erk1,2. Calu3 cells were serum cultured with Corynebacterium diphtheriae toxin @ 10 μg/ml, 25 μM GM6001, 2.5 μM TAPI, 100 μM H2O2, or an equivalent volume of DMSO for 1 hour before lysates were prepared and Western blotted. (E) EGFR neutralizing antibodies blocked phosphorylation in H1975 NSCLC cell line. LAI at 12.5 μg was added to H1975 cells for 18 hours. DMSO or 1 μM AG1478 was added for 1 hour. Lysates were prepared for SDS-PAGE and Western blotting with anti-phospho-EGFR(Y-992) and anti-phospho-EGFR (Y-845) or anti-actin. Caco-2 cells (ATCC #HTB-37, human colorectal adenocarcinoma) served as positive controls for the TACE (ADAM 17) inhibitors, GM6001 and TAPI [35] (data not presented).