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. 2020 Dec 3;3:730. doi: 10.1038/s42003-020-01474-3

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© The Author(s) 2020

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 — a, b Y2H assay revealing the region of PSY1 that interacts with PPSR1. a Schematic illustration for full-length PSY1 protein and the truncated forms used in Y2H analysis. Numbers indicate the positions of the first and last amino acid in the sequences. cTP, chloroplast transit peptide. b The PPSR1 fused with the binding domain (BD) of GAL4 (BD-PPSR1) and the truncated PSY1 fused with the activation domain (AD) of GAL4 were co-expressed in yeast. The transformants were selected on SD/-Leu/-Trp (-LW) and SD/-Leu/-Trp/-His/-Ade (-LWHA) with or without X-α-gal. c LCI assay revealing the interactions between PSY1 and PPSR1. The PPSR1 fused with the C-terminus of LUC (cLUC-PPSR1) was co-expressed with the PSY1 fused with the N-terminus of LUC (PSY1-nLUC) in tobacco (Nicotiana benthamiana) leaves. Scale bar, 1 cm. d Semi-in vivo pull-down assay revealing the interactions between PPSR1 and PSY1. The recombinant MBP-PPSR1 and MBP (negative control) were mixed with PSY1-HA expressed in tobacco leaves, and incubated with anti-HA agarose. The eluted proteins were detected by immunoblot using anti-MBP and anti-HA antibodies, respectively. IB, immunoblot. e Co-IP assay revealing the interactions between PSY1 and PPSR1. The Flag-PPSR1 and PSY1-HA fusion proteins were co-expressed in tobacco leaves. f Subcellular colocalization of PSY1 and PPSR1. The PSY1-eGFP and PPSR1-mCherry fusion proteins were transiently co-expressed into tobacco leaves. The tobacco leaves expressing eGFP or mCherry were used as the negative control. Scale bars, 20 µm. g Ubiquitination assay of PSY1. The Agrobacteria carrying 35S::PSY1-HA, 35S::Flag-ubiquitin (Ub), and 35S::PPSR1 constructs were infiltrated into the tobacco leaves. For (e) and (g), the total proteins were extracted from the infected leaves treated with MG132 and incubated with anti-HA agarose to enrich PSY1-HA. The eluted proteins were subjected to immunoblot using anti-Flag and anti-HA antibodies, respectively. The red arrowhead indicates the predicted Flag-PPSR1. The black arrowhead refers to heavy chain of antibody (IgG_H). Blue asterisks refer to nonspecific bands. IB, immunoblot; (Ub)_n, polyubiquitin chain.