p37 peptide treatment induces HER2 degradation.

<p>(A) Flow cytometric analysis of HER2 levels in SK-BR-3 cells treated with the indicated concentration of p37 for 8 h. HER2-low expressing T47D cells served as a control. (B) IF staining of HER2 in fixed and permeabilized SK-BR-3 cells treated with 20 μg/ml of p37 for 8 h. Scale bar, 40 μm. (C) Immunoblot detection of HER2 in SK-BR-3 cells treated with the indicated concentrations of p37 for 8 h. (D) SK-BR-3 cells were transfected with gp96 or control siRNA for 48 h. Cells were then treated with 20 μg/ml of p37 or control peptide for additional 8 h. Cell lysates were subjected to Western blotting. Hsp 90α, Hsp90β and α-tubulin served as controls. (E) Immunoblot detection of HER2 in SK-BR-3 cells treated with 20 μg/ml of p37 or control peptide along with 100 μg/ml of cycloheximide for the indicated times. (F) Real-time PCR analysis of gp96 and HER2 mRNA levels in SK-BR-3 cells treated with p37 or control peptide. (G) SK-BR-3 cells were incubated with 50 μM MG-132 and the indicated concentrations of p37. HER2 was immunoprecipitated and subjected to immunoblot with anti-ubiquitin antibody. (H) SK-BR-3 cells grown on coverslips were treated with 100 nM Bafilomycin A1 for 1 h. Cells were then treated with the indicated concentrations of p37. Cells were then fixed and stained for HER2 (green) and LAMP-1 (red). Scale bar, 40 μm. Results are presented as means ± SD from three independent experiments.