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. 2018 Nov 22;9:1343. doi: 10.3389/fphar.2018.01343

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Copyright © 2018 Ye, Duan, Deng, Wang, Chen, Cui, Sun, Zhang, Du, Gu, Lin and Tang.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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DENND1A and Grb2 combine with each other. (A) BGC-823 cells lysates were immunoprecipitated with an anti-Grb2 antibody or (B) anti-DENND1A antibody, and then both unprocessed lysates (Input) and immunoprecipitates were analyzed by western blot with indicated antibodies. (C) HEK-293T cells transiently transfected with Flag-Grb2 and HA-DENND1A were immunoprecipitated with an anti-HA antibody or anti- Flag antibody, and then both unprocessed lysates (Input) and immunoprecipitates were analyzed by western blot with indicated antibodies. (D) GST-Grb2 was incubated in vitro with lysates of HEK-293T cells transfected with or without HA-DENND1A, and coprecipitation of DENND1A with GST-Grb2, bound to glutathione-beads, was analyzed by western blot. GST, GST alone used as a control. (E) BGC-823 cells expressing Flag-Grb2 and HA-DENND1A were cultured on coverslips and fixed and double-stained with anti-HA and anti-Flag antibody. (F) Original colors were Flag-Grb2, green; HA-DENND1A, red; and nuclei, blue. Merged pictures represent the composite of all channels, with yellow regions indicative of colocalization. Scale bar, 10 μm. All experiments were repeated at least three times.