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. 2024 Oct 15;15:8887. doi: 10.1038/s41467-024-53277-3

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 2 — a–c Interface I and II mediating the binding of two 14-3-3γ protomers to TLE6 in the SCMC. Interface I (b, marked in red box) and Interface II (c, marked in blue box) show the interactions of two 14-3-3γ protomers with TLE6 pS139 and pS209, respectively. Purple triangles indicate TLE6 pS139 and pS209, respectively. pS, phosphorylated serine. Single components are colored and labeled as denoted. d HEK-293F cells were co-transfected with expression vectors harboring Flag-tagged MATER (or a blank vector for negative control), Strep-tagged FLOPED, HA-tagged 14-3-3γ and either Strep-tagged TLE6, TLE6_S139A mutant, TLE6_S209A mutant, TLE6_S223A mutant, TLE6_S139/209A double-site mutant, or TLE6_{S139/209/223A} triple-site mutant as denoted. 60 h after transfection, cell lysates before (Input) or after immunoprecipitation with anti-Flag affinity agarose gel were immunoblotted for HA-tagged 14-3-3γ, Flag-tagged MATER, Strep-tagged FLOPED and Strep-tagged TLE6 (or mutants). e Table for binding affinity (K_d) between His-tagged 14-3-3γ and TLE6 wildtype peptides (TLE6 (133–144) or TLE6 (203–214)) or phosphorylated peptides (TLE6 (133–144) pS139, TLE6 (203–214) pS209, or TLE6 (217–228) pS223). The data represent the mean ± S.D. (standard deviation) of 3 independent experiments. pS, phosphorylated serine. UD, undetectable. The minus symbol “-” indicates that the peptide TLE6 (217–228) was undetermined due to insolubility. f Schematic overview of mouse zygotes cultured for 24, 48, 72, and 96 h after co-microinjection with mCherry-Trim21 mRNA + isotype IgG + mRNA solvent (control), mCherry-Trim21 mRNA + anti-TLE6 antibody + mRNA solvent (T1), mCherry-Trim21 mRNA + anti-TLE6 antibody + Tle6 mRNA (T2), or mCherry-Trim21 mRNA + anti-TLE6 antibody + Tle6_S139/209A mRNA (T3). The validation for the loss of TLE6 was presented in Supplementary Fig. 7b. The proportions of embryos on different stages were calculated. N = 3 independent experiments. The data are presented as the mean ± S.D.