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. 2021 Sep 9;12:5248. doi: 10.1038/s41467-021-25523-5

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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. 5 — a Analysis of levels of recombinant GST-EFR3A, as detected by immunoblot with an anti-EFR3A antibody, that co-immunoprecipitates (CO-IP) with recombinant His₁₀-KRAS^G12V (preloaded with GTPγS) using an anti-KRAS antibody, in the presence of increasing levels of the RBD of BRAF, detected with and a BRAF-specific antibody. INPUT levels are shown as loading controls. b–e A (b) schematic of GST-EFR3A constructs used (FL: EFR3A base construct lacking 1-107 residues, C: C-terminal fragment, Δ: C-terminal truncations of FL) and (c, d) the levels and (e) quantification thereof of the indicated recombinant GST-EFR3A proteins, as detected by immunoblot with an EFR3A-specific antibody, co-immunoprecipitating (CO-IP) with recombinant His10-KRAS^G12V (preloaded with GTPγS) using a KRAS-specific antibody. INPUT and IP serve as loading controls. GST serves as a negative CO-IP control. f, g Analysis of the (f) levels and (g) quantification thereof of the endogenous EFR3A, as detected by immunoblot with an EFR3A-specific antibody, co-immunoprecipitating (CO-IP) with FLAG-KRAS^G12D using a FLAG-specific antibody when transiently expressed in 293T cells. h Levels of endogenous EFR3A bound to endogenous active KRAS, as assessed by RBD pull-down assay in AsPC-1 cells, as assessed by incubating AsPC-1 cell lysate with recombinant GST-RAF-RBD protein, followed by immunoblot with a KRAS-specific antibody to detect active KRAS and an EFR3A-specific antibody to detect endogenous EFR3A. INPUT and IP levels are shown as loading controls. a, c–h Representative of 3 biological replicates. Replicate experiments and full-length gels are provided in Supplementary Fig. 12. Significance values calculated by one-sided student’s t test for (e, g): ***p < 0.001. Specific p values for (e) are 0.00052 (FL), 0.00035 (C), 0.00011 (Δ1), 0.00014 (Δ2), 0.00056 (Δ3), and 0.00034 (Δ4) and (g) is 0.00087.