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. 2021 Feb 10;12:920. doi: 10.1038/s41467-021-21159-7

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© The Author(s) 2021

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. 2 — a IC₅₀ values of radiolabeled kinase assay for WT RAF and Class 1 and 2 BRAF mutants (mean, n = 2 biologically independent experiments). Plotted values shown in Table 1. b Purified protein ternary complex assay. GST-VBC (VHL, Elongin B, Elongin C) is immobilized on glutathione beads and incubated with DMSO, SJF-0661 (500 nM) or increasing concentrations of SJF-0628 and purified full length-BRAF to observe VBC:PROTAC:BRAF ternary complex. c Quantification of 2b with respect to 1% input (mean ± SD, n = 3 biologically independent samples). Replicates shown in source data; WT = black circles, V600E = blue circles. d Cell lysate based ternary complex assay (as described in b) but using NIH3T3 cell lysates (doxycycline 800 ng/mL) containing V5-BRAF^WT or V5-BRAF^V600E as input. e Quantification of 2d with respect to 1% input (mean ± SD, n = 3 biologically independent samples). Replicates shown in source data. WT = black circles, V600E = blue circles. f NIH3T3 cells expressing indicated V5-BRAF treated with DMSO or 1 µM SJF-0628 for 1-hour followed by immunoprecipitation of V5-BRAF. g NanoBRET ternary complex assay. HEK293T cells ectopically expressing NanoLuc-BRAF(donor) and HaloTag-VHL covalently labeled with a HaloTag 618 ligand (acceptor) were treated with DMSO, epimer SJF-0661(1 μM) or indicated concentration of SJF-0628 for 3 h. Data represented as BRET ratio (mean ± SD, n = 4 biologically independent experiments); WT = black circles, V600E = blue circles. h Tandem Ubiquitin Binding Entities 1 (TUBE1) pull down of tetra-ubiquitinated proteins in NIH3T3 cells expressing indicated V5-BRAF after 1-hour treatment with vehicle or SJF-0628. Immunoblotted for V5-BRAF. Source data are provided as a Source Data file.