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. 2022 Mar 15;13:1352. doi: 10.1038/s41467-022-28978-2

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© The Author(s) 2022

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 GC–MS analysis of products formed by PhBSα and PhBSβ subunits, and their mixture at 1:1 ratio. The response of internal standard in each run was set as 100%. b GC–MS analysis of products formed by PhBS from different hydroxycinnamoyl-CoA substrates. GC-MS chromatograms of in vitro enzymatic assays using various substrates. Purified PhBS (1:1 ratio between α and β subunits) was incubated with benzoyl-CoA and its structural analogs including cinnamoyl-CoA, para-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA. Formation of cinnamaldehyde and coniferaldehyde by purified PhCCR1 was used as a positive control. Shown are combined EICs of mass units 106 (benzaldehyde), 128 (internal standard), 131 (cinnamaldehyde), and 178 (coniferaldehyde). The response of internal standard in each run was set as 100%. IS, internal standard. c Pull-down analysis of PhBSβ-His binding to MBP-PhBSα. Purified MBP-PhBSα was incubated with bacterial lysate of pET32b empty vector (EV) or pET32b expressing PhBSβ-His, protein complex was purified using Amylose resin and analyzed by SDS–PAGE. (*) indicates the position of MBP-PhBSα; triangle indicates the position of PhBSβ-His. The experiment was repeated three times with similar results. d Pull-down analysis of PhBSα binding to PhBSβ-His. Purified MBP-PhBSα was digested with Factor Xa protease, incubated with purified PhBSβ-His, the complex was purified again using Ni-NTA column and analyzed by SDS-PAGE. (*) indicates the position of MBP-PhBSα; (**) indicates the position of free MBP tag; triangle represents the position of PhBSβ-His and untagged PhBSα, as indicated by BS activity. The experiment was repeated three times with similar results. e Y2H detection of PhBSα and PhBSβ interactions. Yeast cells harboring different combination of AD and BD were spotted at increasing dilutions on nonselective (-leu/-trp) and selective medium (-leu/-trp/-his). AD, activation domain of pGAD-T7 and BD, DNA-binding domain of pGBK-T7, to which petunia BSα and BSβ were fused; EV empty vector. At least 10 colonies from each combination were tested on selective medium with same results.