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. 2023 Feb 28;14:947. doi: 10.1038/s41467-023-36496-y

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a, b Depletion of HER2 and EGFR surface receptor levels by treatment with BS4 and EGF, respectively. SkBr3 cells were incubated for 10 min with BS4 or EGF, and ± CytochalasinD (CytoD) followed by cell-surface biotinylation and concentration on Streptavidin beads (surface). Samples were analysed by immunoblot for HER2, EGFR and HER3, TfR as negative controls (a). A quantitation of the results is shown in (b) means ± SD, n = 4 independent experiments, ns (non-significant) P > 0.05, **P = 0.0051, ***P = 0.0009, ***P < 0.0001; two-way ANOVA with Sidak’s multiple comparison test. c, d BS4 clusters HER2 but no other receptors in the plasma membrane resulting in HER2-specific endocytosis. SkBr3 cells were incubated with HER2-specific, dylight650-labelled biparatopic antibody BS4 for 2.5 min. After fixation, total receptor tyrosine-protein kinase ErbB-2 (HER2), receptor tyrosine-protein kinase ErbB-3 (HER3), epidermal growth factor receptor (EGFR), sodium/potassium-transporting ATPase Alpha1 (Na/K-ATPase) and Transferrin-receptor 1 (TfR) in cell sections were stained and samples were analysed by confocal microscopy. The spot overlap of fluorescent signal for BS4 and respective receptors is quantified in (d); means ± SD, n = 6 randomly chosen fields of view with a total of at least 50 cells per condition, ***P < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. e Specific aggregation of HER2 receptor by BS4. SkBr3 cells were incubated with BS4 for indicated times and samples lysed in the low detergent buffer. Samples were spun to separate soluble (Sup) from insoluble/aggregated proteins (Pellet) and immunoblotted for indicated cell-surface receptors. f BS4 specifically endocytoses HER2 receptor. After surface biotinylation, cells were incubated with BS4 for indicated times. Protein was fully solubilised (see “Methods” for protocol) and endocytosed biotinylated proteins concentrated on Streptavidin beads (uptake). Samples were assayed by immunoblot for the HER2, HER3, EGFR, Na/K-ATPase and TfR. Scale bars: 10 µm. Source data are provided as a Source Data file.