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. 2023 Oct 28;14:6898. doi: 10.1038/s41467-023-42618-3

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© The Author(s) 2023

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. 4 — a Interactions between RTFL18 and BSK3/BSK6 were detected by luciferase complementary imaging (LCI). b Interactions between RTFL18 and BSK3/BSK6 were detected via bimolecular fluorescence complementation (BiFC) assays. YFP, YFP signal; Bright, bright field; Merge, merged image of YFP signal with bright field. Scale bars represent 50 μm. c, d Interactions between RTFL18 and BSK3/BSK6 were detected via semi-in vivo pull-down assay. e Interaction between RTFL18 and BSK3/BSK6 were detected via in vitro pull-down assay. f Interactions between Flag-RTFL18 and BSK3/6-GFP were detected via coimmunoprecipitation (CoIP) assay in N. benthamiana leaf cells. g Interactions between Flag-RTFL18 and BSK3/BSK6-GFP were detected via coimmunoprecipitation (CoIP) assay in Arabidopsis. h The BSK3/RTFL18 complex was predicted with AlphaFold-Multimer. The N-terminal domain (NTD) and C-terminal domain (CTD) of BSK3 are green yellow and cyan, respectively. RTFL18 is green. i The hydrophobic interactions between BSK3 and RTFL18. j Hydrogen bond interactions between BSK3 and RTFL18. k The hydrogen bond and electrostatic interactions between BSK3 and RTFL18. l Interactions between different forms of RTFL18 and BSK3 were detected via in vitro pull-down assays. 6His-RTFL18 WT (wild type), M1 (I39A, M43A, L47A), M2 (I40A, H50A, D51A), and GST-BSK3 were expressed in and purified from E. coli. Anti-GST antibodies were used to pull down GST-BSK3. Both the immunoprecipitated fractions and inputs were analyzed by immunoblotting with an anti-His and anti-GST antibodies, respectively. m Interactions between different forms of BSK3 and RTFL18 were detected via in vitro pull-down assays. GST-BSK3 WT (wild type), M1 (E368A, K385A, R393A), M2 (W374A, M378A, L382A), M3 (E368A, K385A, R393A, T374A, M378A, L382A) and His-RTFL18 were expressed in and purified from E. coli. Anti-His antibodies were used to pull down His-RTFL18. In b–g and l, m, each experiment was repeated three times with similar results. Source data are provided as a Source Data file.