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. 2015 Apr 21;10(4):e0124647. doi: 10.1371/journal.pone.0124647

Fig 1. gp96 conformational changes are required for facilitating HER2 dimerization.

Fig 1

(A) 10 μM of purified gp96 was treated with 100 μM of p37 (444–480 aa) or control (61–100 aa) peptides and subjected to trypsin digestion at the indicated pH values. BSA served as a control. (B) ELISA analysis of interaction between gp96 and p37 or control peptides. (C) Flow cytometric analysis of cell surface levels of gp96. (D) SK-BR-3 and T47D cells were cultured in presence of FITC-labeled p37 for 30 minutes, and then stained by immunofluorescence (TRITC) using an anti-gp96 antibody. Scale bar, 40 μm. (E-F) SK-BR-3 cells were treated with 20 μg/ml of p37 or control peptide for 30 min. Cell lysates were immunoprecipitated with anti-HER2 antibody (E). SK-BR-3 cells were pre-treated with EGF (50 ng/ml) or β-heregulin (100 ng/ml) for 15 min, and then cells were treated and analyzed as in E (F). Numbers below the blot indicates quantification shown on Western blot after normalization to HER2. (G) Western blot assay of cell lysates of SK-BR-3 cells treated with 20 μg/ml of p37 or control peptide for 8 h. The ratios of P-AKT to AKT and P-ERK to ERK were calculated, and the values were shown. The ratios in control peptide-treated cells were arbitrarily taken as 1.0.