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. 2018 Feb 27;9:851. doi: 10.1038/s41467-018-03141-y

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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 — Competition of the Gγ proteins in interacting with RGB1. a Interactions of GS3, DEP1 and GGC2 with RGB1 using yeast two-hybrid assay. GS3 interacts with RGB1 through the OSR domain but not the C-terminal cysteine-rich domain (TV). OSR: OSR domain of GS3, amino acid 1-94; TV: cysteine-rich domain of GS3, amino acid 95-231. AD: GAL4 activation domain; BD: GAL4 binding domain. -LW: selective medium lacking Trp and Leu; -LWHA: selective medium lacking Trp, Leu, His and Ade. b Interactions of GS3, DEP1 and GGC2 with RGB1 using BiFC assay. Bar = 20 μm. c Interactions of GS3, GGC2 and DEP1 (fused to C-terminal fragment of firefly luciferase) with RGB1 (fused to N-terminal fragment of firefly luciferase) using luciferase activity assay. Rice protoplasts with transient expression of RGB1-nLuc plus cLuc-DEP1, cLuc-GGC2, cLuc-GS3-1, and cLuc-GS3-4, but not the C-terminal cysteine-rich domain (TV), show high luciferase activity relative to the control. Empty vectors of cLuc plus RGB1-nLuc, and nLuc plus cLuc-DEP1, cLuc-GGC2, cLuc-GS3-1, and cLuc-GS3-4 were used as the negative control. Data are normalized to the internal control 35 S::REN. Values are given as mean ± SEM (n = 3). Different letters indicate significant differences ranked by the LSD test (P < 0.05). d Interactions of DEP1 and GGC2 with RGB1 in the background of ZH11, GS3-1OE, and GS3-4OE using luciferase activity assay. RGB1-nLuc plus cLuc-DEP1, and RGB1-nLuc plus cLuc-GGC2 are transiently expressed in the protoplasts of ZH11, GS3-1OE, and GS3-4OE, respectively. Empty vector of cLuc plus RGB1-nLuc is used as the negative control. Data are normalized to the internal control 35 S::REN. Values are given as mean ± SEM (n = 3). Different letters indicate significant differences ranked by the LSD test (P < 0.05). e Yeast three-hybrid assay for protein interactions of DEP1/GGC2 and GS3 with RGB1. The interaction of DEP1/GGC2 and GS3 with RGB1 is analyzed using fusions with AD (AD-DEP1, GGC2 or GS3) and BD (BD-RGB1). Empty vectors are used as a negative control. Quantitative analysis of interactions by β-galactosidase assay is shown. Data for all the assays are shown as mean ± SD (n = 3)