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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. 4 — a–d Analysis of (a, c) the levels and (b, d) quantification of biotin labeling of FLAG-EFR3A expressed in 293T cells by ectopic myc-BirA*-KRAS wild-type (WT), G12V, or S17N or by myc-BirA alone as a control, as assessed by streptavidin affinity capture (IP) of biotinylated proteins and immunoblotted with FLAG- and myc-specific antibodies. INPUT levels are shown as loading controls. e–h Analysis of (e, g) the levels and (f, h) quantification of recombinant GST-EFR3A detected by immunoblot with an EFR3A-specific antibody that co-immunoprecipitates (co-IP) WT or G12V recombinant His₁₀-KRAS using a KRAS-specific antibody, and (g, h) preincubated with buffer, GDP, or GTPγS. INPUT levels are shown as loading controls. IgG serves as a negative control. i, j Analysis of (i) the levels and (j) quantification of recombinant His₁₀-KRAS^G12V (preloaded with GTPγS) detected with a KRAS-specific antibody, that associates with recombinant GST-EFR3A versus control GST by far western. INPUT levels are shown as loading controls. k, l Analysis of (k) the levels and (l) quantification of GST-EFR3A detected with an EFR3A-specific antibody, that associate with recombinant wild-type (WT) versus His₁₀-KRAS^G12V by far western. INPUT levels are shown as loading controls. m–r Analysis by (m, o, q) size-exclusion chromatography (SEC) profile and (n, p, r) immunoblot of the indicated fractions of recombinant (m, n) GST-EFR3A (peak 15.48: GST-EFR3A, o, p recombinant His₁₀-KRAS^G12V preloaded with GTPγS (peak 18.49: His₁₀-KRAS^G12V), and (q, r) a complex of recombinant GST-EFR3A and His₁₀-KRAS^G12V preloaded with GTPγS (peak 15.29: complex). a–k Representative of 3 biological independent samples tested in 3 independent experiments. m–r 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 (b, d, f, h, j, l): **p < 0.01 and ***p < 0.001. Specific p values for (b) is 0.00089, d are 0.00074 (G12V) and 0.00014 (S17N), f is 0.00058, (h) are 0.00048 (WT^GDP), 0.00015 (WT^GTP), 0.00031 (G12V^GDP), and 0.00028 (G12V^GTP), j is 0.00067, and l is 0.0069.