Additional file 3: of Polyfunctional anti-human epidermal growth factor receptor 3 (anti-HER3) antibodies induced by HER3 vaccines have multiple mechanisms of antitumor activity against therapy resistant and triple negative breast cancers

Figure S2. Flow cytometric detection of cell surface EGFR/HER2/HER3 expression after treatment with HER3-VIA, trastuzumab, cetuximab and lapatinib. To assess the internalization of EGFR family receptors, SKBR3 cells, positive for EGFR/HER2/HER3, were incubated with HER3-VIA, GFP-VIA (1:100 dilution), trastuzumab (1 μM), cetuximab (1 μM), or lapatinib (1 μM) for 3 h or 24 h. Cells were harvested using cell-dissociation buffer and stained with PE-conjugated anti-EGFP (clone 5E10D3, Novus Biologicals), anti-HER2 (clone Neu 24.7, BD Bioscience) or anti-HER3 antibody (Clone 1B4C3, BioLegend) and acquired by LSRII flow cytometer. Isotype control mouse IgG was used as negative control staining and is shown as filled gray histograms. Experiments were performed four times for EGFR expression analysis and twice for HER2 and HER3, and representative histograms are shown. Median fluorescence intensities (MFIs) of reagent-treated cells were compared to untreated control cells and ratios (MFI of treated/MFI of untreated) were calculated in each experiment. The averages of ratios are shown in each histogram. (PDF 732 kb)