Figure 3. Inhibition of EGFR activation and downstream signaling using H-helix-derived peptides.

a) PEG₃-conjugated H-helix based peptides were tested for their ability to inhibit EGFR phosphorylation as a marker of EGFR inhibition using MDA-MB-231 cells. The ratio of phosphorylated EGFR (pY1068) to the loading control α-tubulin was measured by western blot analysis. Peptides 14 and 17 were found to downregulate phosphorylation. Densitometry is shown in the graph above the western blots using 3 independent experiments performed in triplicate. b) A concentration-dependent cell-based assay was performed using Peptide 17 (EHBI2) and its corresponding scramble control peptide. EHBI2 demonstrated dose-dependent inhibition of EGFR activation while its scramble control had no effect at the same concentrations. Densitometry data represents n=3 experiments performed in triplicate. c) To assess effects on downstream signaling of EGFR, total Akt, phosphorylated Akt (S473) and GAPDH were measured by western blotting. Densitometry data was measured from a triplicate experiment. EHBI2 caused downregulation of Akt phosphorylation, but not its scramble control. One-way ANOVA and a Tukey’s post hoc test (95% confidence interval) were performed on all densitometry data sets. The p-values are as follows: *** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05 as compared to the positive control (+EGF). Error bars represent standard error of the mean.