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. 2019 Sep 23;9:13703. doi: 10.1038/s41598-019-50275-0

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© The Author(s) 2019

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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Phosphorylation of ADD1 at T724 is important for lamellipodia formation. (a) MDA-MB-231 cells were infected with lentiviruses expressing shRNAs specific to ADD1 (sh-ADD1) or luciferase (shLuc). FLAG-ADD1 or its mutants were re-expressed in the ADD1-depleted cells (sh-ADD1/FLAG-ADD1). The cells were serum-starved for 18 h and treated with 40 ng/ml EGF for 15 min. An equal amount of whole cell lysates was analyzed by immunoblotting with the indicated antibodies. (b) The cells as described in (a) were plated on collagen-coated coverslips, serum-starved, and treated with (+) or without (−) 200 ng/ml EGF for 6 h. The cells were fixed, stained for cortactin and F-actin, and visualized with a Zeiss ApoTome2 system. Representative micrographs are shown. Bars, 20 μm. (c) The percentage of cells with lamellipodia in the total number of counted cells was measured (n ≥ 200). Values (means ± s.d.) are from three independent experiments. **P < 0.01; ***P < 0.001.