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. 2018 Sep 17;9:3784. doi: 10.1038/s41467-018-05913-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. 3 — BRAF colocalizes with FREE1 in MVB/PVCs and interacts with ESCRT-I component Vps23. a BRAF-mRFP shows plasma membrane and intracellular punctate dots. BRAF-mRFP-labeled punctae partially colocalize with GFP-FREE1 in Arabidopsis root epidermal cells. Arrows indicate colocalized dots. Colocalization relationship was calculated by Pearson–Spearman correlation. Scale bar, 10 μm. b GFP-FREE1 and BRAF-mRFP colocalize to wortmannin (Wort)-induced enlarged MVB/PVCs. Seedlings expressing GFP-FREE1 and BRAF-mRFP as shown in (a) were treated with Wort for 40 min before imaging. Separated images of each channel in the white outline area are shown on the right side (from top to bottom: GFP, RFP, and merged). Scale bar, 10 μm. c Y2H analysis of the binary interactions of BRAF with FREE1, ESCRT-I component (Vps23A, Vps23B, Vps28A, and Vps37A), ESCRT-III component (SNF7A and SNF7B), or deubiquiting enzyme AMSH1. Transformed yeast cells were grown on either synthetic complete medium lacking leucine and tryptophan (with histidine, +His) as a transformation control, or synthetic complete medium lacking leucine, tryptophan, and histidine (without histidine, −His) for interaction assays. 3-AT is used to suppress the background self-activation of the BD genes. d In vitro binding assays of recombinant MBP (lane 1), MBP-Vps23A (lane 2), or MBP-Vps28A (lane 3) with BRAF. Anti-BRAF and anti-MBP antibodies were used to detect bead-retained material. Arrowhead indicates BRAF protein pulled down by Vps23A. e FRET analysis of the colocalized punctae between BRAF-YFP and the two Vps23 homologs (Cerulean-Vps23A and Cerulean-Vps23B) or Cerulean-Vps28A. FRET efficiency was quantified by using the acceptor photobleaching approach on the right bottom. For each group, 10 individual protoplasts were used for FRET efficiency quantification and statistical analysis. Error bars are the S.D. of FRET efficiency. f Immunoprecipitation (IP) assay shows association between BRAF and FREE1 with Vps23A. Arabidopsis protoplasts expressing EYFP (lane 1) or EYFP-Vps23A (lane 2) with 3 × Myc-FREE1 and BRAF-4 × HA were subjected to protein extraction and IP with GFP-trap followed by immunoblot with indicated antibodies. Arrowhead indicates BRAF and FREE1 proteins immunoprecipitated by Vps23A