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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. 4 — BRAF competes with FREE1 for ESCRT-I component Vps23 binding. a Domain structures of BRAF, FREE1, and Vps23A. UEV ubiquitin E2 variant, PRR proline-rich region, CC coiled-coil, SB steadiness box. b Y2H analysis of the binary interactions between BRAF (left) or FREE1 (right) with different domain deletions of Vps23A. c In vitro binding assays of recombinant BRAF (lanes 1 and 2) or FREE1 (lanes 3 and 4) with MBP (lanes 1 and 3) and MBP-Vps23A(CC) (lanes 2 and 4). d Competition assay. Binding assay of MBP-Vps23A with 30 pmol of BRAF was performed in the presence of 10, 30, 100, or 300 pmol of FREE1. Binding assay of MBP-Vps23A with constant FREE1 was also performed in the increasing amounts of BRAF or BRAF(A330V). The pull-down efficiency is shown on the right. The amount of proteins on the sepharose was compared with that of the control pair (as 100%) of MBP-Vps23A/FREE1 (lane 7) or MBP-Vps23A/BRAF (lane 11). The intensity of the pull down was normalized by the MBP-Vps23A intensity. Error bars are the S.D. from three independent experiments. e Immunoprecipitation (IP) analysis between 3 × HA-BRAF or 3 × HA-BRAF(A330V) and Vps23A. Arabidopsis protoplasts expressing EYFP (lanes 1 and 3) or EYFP-Vps23A (lanes 2 and 4) with 3 × HA-BRAF (lanes 1 and 2) or 3 × HA-BRAF(A330V) (lanes 3 and 4) were subjected to protein extraction, followed by immunoprecipitation (IP) using GFP-Trap agarose beads and subsequent immunoblot analysis on eluted proteins with indicated antibodies. f In vitro pull-down assays of BRAF or BRAF(A330V) with Vps23A. Recombinant His-Sumo-BRAF (lanes 1 and 2) or His-Sumo-BRAF(A330V) (lanes 3 and 4) was incubated with either MBP (lanes 1 and 3) or MBP-Vps23A (lanes 2 and 4) for 1 h at 4 °C and subjected to immunoblot analysis. g Quantification analysis of binding efficiency in IP analysis and in vitro pull-down assays. The amount of binding protein in BRAF(A330V)-Vps23A was compared with that of the control pair of BRAF-Vps23A (as 100%). The intensity of the immunoblot was normalized by the input. Error bars represent the S.D. from three independent immunoblot results. **P < 0.01 in Student’s t-test