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. 2018 Oct 29;8:15981. doi: 10.1038/s41598-018-34425-4

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© The Author(s) 2018

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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SHP2-binding activity of Hp_TH2099 CagA. (a) AGS cells were transiently transfected with an ABC’-CagA-FLAG vector, an ABC-CagA-FLAG vector, or an empty control vector (SRα). Cell lysates were immunoprecipitated with an anti-FLAG antibody and the immunoprecipitates were subjected to immunoblotting analysis with the indicated antibodies. TCL: total cell lysate. IP: immunoprecipitate. Full-length blots are presented in Supplementary Figure S12. (b) CagA-binding interface of the SHP2 N-SH2 domain based on the data described previously³⁹. Red-to-blue represents acidic-to-basic on the surface of the SHP2 N-SH2 domain. A dot circle in yellow indicates the region that interacts with the pTyr + 5 residue of the EPIYA-C segment. A dot circle in black indicates the region corresponding to the pTyr-binding pocket. (c) AGS cells were transiently transfected with an ABC’-CagA-FLAG vector, an ABC’^LP-CagA-FLAG vector, an ABC-CagA-FLAG vector, an ABC^PL-CagA-FLAG vector, or an empty control vector (SRα). In the ABC’^LP-CagA-FLAG mutant, the ELIYA sequence in the EPIYA-C segment of ABC’-CagA-FLAG was replaced by the EPIYA sequence. In the ABC^PL-CagA-FLAG mutant, the EPIYA sequence in the EPIYA-C segment of ABC-CagA-FLAG was replaced by the ELIYA sequence. Cell lysates were immunoprecipitated with an anti-FLAG antibody and the immunoprecipitates were subjected to immunoblotting analysis with the indicated antibodies. Full-length blots are presented in Supplementary Figure S12. (d) The morphology of AGS cells transfected with an ABC’-CagA-FLAG or an ABC-CagA-FLAG vector was examined under a microscope at 17 hours after transfection. Scale bars: 100 µm (left). Percentages of cells showing the hummingbird phenotype were calculated and statistically analyzed by using Student’s t-test. Error bars represent ± S.D. (n = 3), **p < 0.01. (right).