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. 2024 Mar 4;15:1982. doi: 10.1038/s41467-024-45805-y

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© The Author(s) 2024

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 licence, and indicate if changes were made. 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/4.0/.

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Fig. 6 — a–c Analyses of T and B lymphocytes of CMV-Cre; Ctps2^flox/flox (Ctps2^ko/ko) and control littermate (Ctps2^wt/flox, Ctps2^flox/flox and Ctps2^wt/ko) animals. a, b Analysis of splenic enriched T cells activated by anti-CD3/CD28 plus IL-2 for 3 days. a, c Analysis splenic purified B cells activated with LPS plus IL-4 for 3 days. a Immunoblots for CTPS1, CTPS2 and Actin expression in lysates of activated T cells (left panel), activated B cells (central panel) and fibroblasts recovered from ear skin (right panel). Molecular weights in kDa on the left. Blots represent two pooled, independent experiments. b Representative proliferation dot plot profiles (left panels) from FACS analysis of enriched T cells labelled with cell trace violet (CTV) and stained for CD25, as an activation marker. Graph bars (right panels) corresponding to proliferation indexes calculated from histogram profiles. c Representative proliferation dot plot profiles (left panels) from FACS analysis of B cells labelled with cell trace violet (CTV) and stained for IA/IE, as an activation marker. Graph bars (right panels) corresponding to proliferation indexes calculated from histogram profiles. d–f Analysis of from CD4-Cre; Ctps1^wt/floxxCtps2^wt/flox (Ctps1^wt/ko, Ctps2^wt/ko) empty black circle, CD4-Cre; Ctps1^flox/floxxCtps2^wt/flox (Ctps1^ko/ko, Ctps2^wt/ko) filled red circle, CD4-Cre; Ctps1^wt/floxxCtps2^flox/flox (Ctps1^wt/ko, Ctps2^ko/ko) filled blue circle and CD4-Cre; Ctps1^flox/floxxCtps2^flox/flox (Ctps1^ko/ko, Ctps2^ko/ko) filled purple circle. d Spleen cell counts (left panel) and percentages of splenic B, T, CD4⁺, CD8⁺ and NK cells calculated from FACS analyses. e Immunoblots for CTPS1, CTPS2 and Actin protein expression in lysates from non-activated or activated splenic T cells, sorted and non-sorted on CD3⁺. Molecular weights in kDa on the left. Blot is representative of two independent experiments. f Representative proliferation dot plot profiles (right panels) from FACS analysis of CD4⁺ (upper panels) and CD8⁺ (lower panels) T cells labelled with cell trace violet (CTV) and stained for CD25 as an activation marker. Graph bars (right panels) corresponding to proliferation indexes calculated from histogram profiles. Non-parametric Matt-Whitney two-tailed test (b) and Unpaired t-tests two-tailed (d, f) were used. Data are presented as the mean ± SD of n = 5 animals per group (b), of n = 8 (controls), n = 6 (Ctps1ko), n = 9 (Ctps2ko) and n = 6 (DKO) animals per group (d) and of n = 7 (controls), n = 5 (Ctps1ko), n = 7 (Ctps2ko) and n = 5 (DKO) animals per group (f). Source data are provided in Source Data file.