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. 2018 Apr 18;9:1522. doi: 10.1038/s41467-018-03884-8

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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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Fig. 5 — BRI1 is required for the phosphorylation of AGB1 and AGG3. a BRI1 kinase domain (BRI1-KD) phosphorylates AGB1 in vitro, but bri1-301 kinase domain (bri1-301-KD) does not. The phosphorylated MBP-AGB1 (pMBP-AGB1), unphosphorylated (MBP-AGB1) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. b BRI1-KD phosphorylates AGG3 in vitro, but bri1-301-KD does not. The phosphorylated MBP-AGG3 (pMBP-AGG3), unphosphorylated (MBP-AGG3) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. c, d BRI1 is required for the phosphorylation of AGB1 in Arabidopsis. Total proteins from pBRI1:BRI1-GFP;35S:Myc-AGB1 (L1) and 35S:GFP;35S:Myc-AGB1 (L2) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads, and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively c. Quantification of the phosphorylated Myc-AGB1 was relative to the immunoprecipitated Myc-AGB1 d. Values are given as mean ± SD (n = 3). **P < 0.01 compared with the value for L2 seedlings by Student’s t-test. e, f BRI1 is required for the phosphorylation of AGG3 in Arabidopsis. Total proteins from pBRI1:BRI1-GFP;35S:Myc-AGG3 (L3) and 35S:GFP;35S:Myc-AGG3 (L4) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively (e). Quantification of the phosphorylated Myc-AGG3 was relative to the immunoprecipitated Myc-AGG3 f. Values are given as mean ± SD (n = 3).**P < 0.01 compared with the value for L4 seedlings by Student’s t-test. g, h BRI1 is required for the phosphorylation of AGB1 in Arabidopsis. Total proteins from 35S:GFP-AGB1 (L5) and 35S:GFP-AGB1;bri1-301 (L6) seedlings were incubated with GFP-Trap-A, and detected using anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively g. Quantification of the phosphorylated GFP-AGB1 was relative to the immunoprecipitated GFP-AGB1 h. Values are given as mean ± SD, (n = 3).**P < 0.01 compared with the value for L5 seedlings by Student’s t-test. i, j BRI1 is required for the phosphorylation of AGG3 in Arabidopsis. Total proteins from 35S:GFP-AGG3 (L7) and 35S:GFP-AGG3;bri1-301 (L8) seedlings were incubated with GFP beads, and detected by immunoblot with anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively i. Quantification of the phosphorylated GFP-AGG3 was relative to the immunoprecipitated GFP-AGG3 j. Values are given as mean ± SD (n = 3).**P < 0.01 compared with the value for L7 seedlings by Student’s t-test