Regulation of cargo‐selective endocytosis by dynamin 2 GTPase‐activating protein girdin AThe endocytic sites for the indicated cargoes were investigated.

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Regulation of cargo‐selective endocytosis by dynamin 2 GTPase‐activating protein girdin AThe endocytic sites for the indicated cargoes were investigated using TIRF microscopy according to the methods described in the Materials and Methods section. Arrowheads indicate vesicles responsible for EGF and integrin β1 internalization at the periphery of the cells. Scale bar, 20 μm. Shown in the right panel is the illustration indicating the endocytic sites for each cargo. See also Supplementary Fig S5A. B, CHeLa cell lysates were subjected to immunoprecipitation by the indicated antibodies, followed by Western blot analysis. The results showed that girdin interacts with EGFR and integrin β1 but not E‐cadherin or TfR. D, EIn vitro binding assays using purified recombinant proteins demonstrated the direct interaction of girdin NT with the cytoplasmic domains of EGFR (EGFRc) (D) and integrin β1 (ITGB1c) (E) but not the extracellular domain of EGFR (EGFRe). In (D), the precipitated GST fusion proteins are indicated by asterisks. F, GThe dose‐dependent competition of EGFR and integrin β1 for the binding of dynamin 2 to the girdin NT domain. GST‐fused girdin NT (3 μg) was incubated with dynamin 2‐His (30 μg) in the presence of increasing amounts of EGFRc‐His (F) or ITGB1c‐His (G). The mixtures of the proteins were precipitated with glutathione Sepharose beads, followed by Western blot analysis. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 33, Issue: 18, Pages: 2098-2112, First published: 24 July 2014, DOI: (10.15252/embj.201488289)