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. 2020 Mar 13;11:1357. doi: 10.1038/s41467-020-15173-4

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© The Author(s) 2020

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. 3 — a Epha2^+/+, Epha2^−/− or Epha2^−/− mESCs (clone C4) stably expressing EPHA2 were cultured in the absence of LIF for 48 h. EPHA2, KLF4, DNMT3B, NANOG and OCT4 levels were determined by immunoblotting. b Epha2^+/+ or Epha2^−/− (clone C4) mESCs were maintained in 2i or differentiated in N2B27 media for 72 or 96 h, respectively, whereupon 10% of cells were replated in 2i. Total alkaline phosphatase staining is represented relative to Epha2^+/+ mESCs. The total number of alkaline phosphatase-positive colonies for Epha2^+/+ and Epha2^−/− mESCs is shown, and also represented relative to Epha2^+/+ mESCs. Data show mean ± SEM (n = 3); statistical significance was determined using unpaired two-sided Student’s t test comparing Epha2^−/− with the Epha2^+/+ control (****P < 0.0001, **P = 0.0016). c Epha2^+/+ or Epha2^−/− (clone C4) mESCs stably expressing EFNA1, along with the respective parental controls, were grown in LIF/FBS, and KLF4, NANOG, EPHA2, EFNA1 and ERK1/2 levels determined by immunoblotting, or EPHA2 immunoprecipitated and pTyr and EPHA2 levels determined by immunoblotting. d Epha2^+/+, Epha2^−/− or Epha2^−/− mESCs stably expressing EPHA2 were differentiated as embryoid bodies for 10 days, and the levels of Fgf5, Brachyury, Mixl and Cer1 mRNA determined by qRT-PCR. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values. The results shown are for technical replicates from two independent experiments, including three Epha2^−/− clones (n = 3); statistical significance at day 4 was determined using unpaired two-sided Student’s t test comparing each group with the Epha2^+/+ control (ns = not significant, *P = 0.0252, **P = 0.0045, ***P < 0.0001). e Epha2^+/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. ppERK1/2, total ERK1/2, STAT3 pY705 and total STAT3 levels were determined by immunoblotting. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting. ppERK1/2 signal was quantified; data show mean ± SD (n = 3); statistical significance was determined using one-sample two-sided t test comparing each group with control, theoretical mean = 1 (ns = not significant, 5 min; *P = 0.0467, 30 min; ***P = 0.0005, 45 min; **P = 0.0014, 60 min; **P = 0.0018). f Epha2^+/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. SHP2 was immunoprecipitated, and SHP2 and EPHA2 levels detected by immunoblotting. Source data are provided as a Source Data file.