A Potential Peptide Therapeutic Derived from the Juxtamembrane Domain of the Epidermal Growth Factor Receptor
(A) SK-N-MC cells were transfected with the intracellular domain (ICD) of EGFR (645–1186) or the ICD of EGFR lacking the entire JXM region (ΔJM) or the JMA region (ΔJMA). Biotinylated peptides at a concentration of 0.1 µM (+) or 0.5 µM (++) were incubated with SK-N-MC cells for 2 hours and precipitated from cellular lysates with streptavidin-coated beads. The resulting bead-precipitates were analyzed by Western blot for the presence of the EGFR constructs. Results are representative of three independent experiments. (B) Streptavidin beads were pre-bound with biotinylated peptides and incubated with transfected SK-N-MC lysates. The non-biotinylated version of TE-64562 was added to compete for binding in lanes 3 and 4. The resulting bead-precipitates and lysates were analyzed by Western blot for the presence of the EGFR constructs. Results are representative of two independent experiments. (C) Serum starved MDA-MB-231 cells were treated with TE-64562 (12.5 and 25.0 µM), an EGFR specific tyrosine kinase inhibitor (TKI, 1.0 µM), Tat (25.0 µM) or vehicle for 30 minutes, followed by EGF treatment (25 ng/mL) for 10 minutes. Cellular proteins were cross-linked using bis(sulfosuccinimidyl) suberate (BS3), cells were lysed and lysates analyzed by Western blot for EGFR. The quantification of the dimer band is shown. The EGFR dimer band 25.0 µM TE-64562 was not detectable (N.D.). Results are representative of three independent experiments.