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. 2022 Dec 29;24(1):136–147. doi: 10.1038/s41590-022-01372-2

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© The Author(s) 2022

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, Total thymocytes from 6- to 8-week-old C57BL/6 female mice were divided into DN, DP, CD4SP and CD8SP populations based on expression of CD4 and CD8α (CD8). b,c, Histograms (b) and quantification (c) of PIP₂ in each thymocyte subset are depicted (n = 4) MFI, mean fluorescence intensity. d, DN cells were divided into four stages (DN1–DN4) based on the expression of CD44 and CD25. e, DN3 cells were subgated into DN3a and DN3b subsets based on intracellular TCRβ (icTCRβ) and surface expression of CD27 assessed by flow cytometry. f,g, Histograms (f) and quantification (g) of PIP₂ in each DN subset were assessed. h, DP thymocytes were subdivided based on amounts of CD5 and TCRβ (DP1 = CD5^loTCRβ^lo, DP2 = CD5^intTCRβ^lo, DP3 = CD5^hiTCRβ^lo, DP4 = CD5^hiTCRβ^int, DP5 = CD5^hiTCRβ^hi). i,j, Histograms (i) and quantification (j) of PIP₂ in each DP subset were assessed and are presented. iso, isotype control. k, Quantification of TCRβ in each DP subset. n = 4. Data are shown as the mean ± s.e.m. and are representative of three independent experiments. P values were determined using an unpaired, one-way analysis of variance (ANOVA) with multiple comparisons.

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