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. 2019 Mar 13;10:1199. doi: 10.1038/s41467-019-09181-2

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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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Fig. 2 — PKS5 enhances the interaction between SOS2 and 14-3-3 proteins. a In vitro pull-down assay showing that PKS5 enhances the interaction between SOS2-JD and 14-3-3λ following incubation with SOS2-JD in kinase buffer containing ATP. JD, SOS2-JD; JD^SA, SOS2-JD^S294A. b In vivo Co-immunoprecipitation assay showing that PKS5 enhances the interaction between SOS2 and 14-3-3 proteins. Myc-SOS2 was extracted with anti-C-Myc agarose from 10-day-old seedlings expressing Pro35S:6×Myc-SOS2 in BigM/pks5-3/pks5-4/Col-0/pks5-1 backgrounds and analyzed by immuno blotting with anti-C-Myc and anti-14-3-3 antibodies. c Luciferase complementation imaging assay showing that co-expression of PKS5/PKS5-3/PKS5-4 in N. benthamiana enhanced the interaction between SOS2 and 14-3-3λ. d Relative fluorescence analysis of (c) by ImageJ. Error bars represent SD; p ≤ 0.05, Student’s t-test; n = 3; significant differences are indicated by different lowercase letters. e, f In vitro kinase assay showing that PKS5 negatively regulates SOS2 activity. Myc-SOS2 was extracted using anti-C-Myc agarose from 10-day-old transgenic seedlings treated with or without 100 mM NaCl for 12 h. His-SCaBP8 (SC8) and MBP were used as the substrates and anti-C-Myc antibody was used for immunoblot analysis. g, h In vivo Co-immunoprecipitation assay showing that PKS5 negatively regulates the phosphorylation status of SCaBP8. Myc-SCaBP8 was extracted from 10-day-old seedlings expressing Pro35S:6×Myc-SCaBP8 in the BigM/pks5-3/pks5-4/Col-0/pks5-1 backgrounds treated or not with 100 mM NaCl for 12 h and analyzed by immunoblotting with anti-C-Myc and anti-phospho-SCaBP8Ser237 (anti-P-SC8) antibodies. i, j SOS recruitment system assays showing that PKS5 inhibits the SOS pathway by phosphorylating SOS2^Ser294 and enhances the interaction between SOS2 and 14-3-3λ. Yeast cells expressing Arabidopsis SOS1 were co-transformed with the indicated plasmids, two positive clones with 3.5 μL of serial five-fold dilutions were grown on AP medium with different concentrations of NaCl to analyze the salt tolerance of yeast strains expressing SOS2, SOS2T168D/Δ308; SOS2^S294D, SOS2T168D/Δ308/S294D. IB immunoblot. CBB Coomassie Brilliant Blue, Autorad autoradiograph. Source data of a, b, and e–h are provided in the Source Data file