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. 2023 Jan 23;14:365. doi: 10.1038/s41467-023-35975-6

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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. 5 — a CTR1 interacts with EBF1 and EBF2, but not EIN3 in yeast-two-hybrid assays. b BiFC assay for full-length wild-type CTR1 and nuclear ethylene signaling proteins in N. benthamiana in the presence of ACC. COP1-interacting protein 8 (CIP8) and bZIP-RFP were used as a negative control and a nuclear subcellular marker, respectively. c Co-immunoprecipitation analysis of CTR1 and EBF2 in N. benthamiana. d Three-day-old dark-grown seedlings were treated with the indicated concentrations of ACC for 2 h, and total protein extracts were used for immunoblotting with anti-EIN3, GFP, and Hsc70 antibodies. e, f ctr1-8 (e) and seedlings expressing GFP-CTR1-8-SV40 NLS in the ctr1-8 mutant (f) expressed higher basal levels of EIN3 than wild-type seedlings. The experiments were repeated at least three times with similar results. g 35S:GFP-ΔNT-gCTR1^ctr1-1 expressed a significantly higher level of EIN3 protein than the wild type after removal of ACC. Seedlings pretreated with 200 µM ACC for 2 h and total protein extracts were harvested at the indicated times after ACC removal. Rel. quantities represent the ratio of the intensity of the EIN3 band to Hsc70 band signals, and these values are expressed relative to the intensity of EIN3/Hsc70 in the wild type with that in time 0 samples, which was set to 1. h ACC dose-response curves for the hypocotyl length of 3-d-old dark-grown wild-type and 35S:GFP-ΔNT-CTR1^ctr1-1 seedlings. Control treatments included no ACC. Error bars, SE (n ≥ 24 seedlings for each ACC concentrations). Two-tailed Student’s t test. i Seedlings expressing GFP-ΔNT-gCTR1^ctr1-1 from its native promoter in ctr1-2 mutant background showed an enhanced ACC response compared to CTR1p:GFP- gCTR1^ctr1-1/ctr1-2 seedlings in the dark. Error bars, SE (n ≥ 46 seedlings for each ACC concentrations). Two tailed Student’s t test. j CTR1 does not phosphorylate EBF2. In vitro kinase assay for purified ΔNT-CTR1 or ΔNT-CTR1^ctr1-1 with EIN2-CEND, EIN3, or EBF2. EIN2-CEND and EIN3 were used as a positive and negative control, respectively. The indicated proteins were incubated together in kinase reaction buffer and then separated by SDS/PAGE, and the incorporated radiolabel was detected by autoradiography. Experiments were repeated three times with the similar results.