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. 2018 Jun 14;9:2341. doi: 10.1038/s41467-018-04719-2

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© The Author(s) 2018

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 — SLC7A5 activity is required for IL-2/IL-12-induced cMyc expression. a NK cells were activated for 18 h with IL-2/IL-12 and the number of protein copies per cell were determined using quantitative proteomic analysis: SLC7A5, SLC7A8, SLC43A1 and SLC43A2. b NK cells were left unstimulated (US) or were stimulated with IL-2/IL-12 for 18 h and Slc7a5 mRNA levels analysed by qPCR. c NK cells were activated with IL-2/IL-12 for 20 h and then switched into media containing cytokines as shown for a further 8 h. Slc7a5 mRNA levels were analysed by qPCR. d Purified NK cells were stimulated with IL-2/IL-12 for 18 h and uptake of ³H-labelled phenylalanine was measured in the presence or absence of the system L competitor BCH (10 mM). e NK cells were stimulated with IL-2/IL-12 for 18 h and the system L blocker BCH (25 mM) was added for the final 30 or 60 min as indicated before immunoblot analysis of cMyc, phosphorylated S6 ribosomal protein on serine 235/6 (pS6) and total S6 ribosomal protein (S6). f Slc7a5^−/− (Slc7a5^flox/flox × Vav-Cre) or WT (Slc7a5^WT/WT × Vav-Cre) NK cells were left unstimulated or were stimulated for 18 h with IL-2/12 before immunoblot analysis of cMyc and β-actin protein expression and qPCR analysis of Slc7a5 mRNA expression. g NK cells were stimulated with IL-2/IL-12 in the presence or absence of leucine for 18 h before immunoblot analysis of cMyc, pS6 and Akt. h Purified NK cells were stimulated with IL-2/IL-12 for 18 h and glutamine uptake was measured using ¹⁴C-labelled glutamine in the presence or absence of BCH (10 mM). i NK cells stimulated with IL-2/IL-12 for 18 h were cultured in the presence or absence of glutamine for 30 or 60 min as indicated, before immunoblot analysis for levels of cMyc and β-actin. j IL-2/IL-12-activated NK cells were cultured for 1 h in IL-2/IL-12 media with or without the amino acids l-glutamine or l-leucine or the addition of rapamycin. Data are mean ± s.e.m of 3 experiments (a–h) or is representative of 2 (f) or 3 individual experiments (e–j). Statistical analysis was performed using a one-way ANOVA with Tukey post test (b–h) or Student's t-test (a); *p < 0.05, **p < 0.01, ***p < 0.005, ns non-significant, ND not detected