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. 2018 Apr 30;9(5):478. doi: 10.1038/s41419-018-0528-7

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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 — a CD8 T cells isolated from peripheral blood of healthy human were transfected with indicated lentiviral particles (wild type Tim-3 and Lnc-Tim3 wild type or Δ226-277). The mRNA levels of LAG3, PRDM1, and PBX3 were assessed by real-time PCR after cells treated with anti-TCR/anti-CD28 beads and PMA-ionomycin. (n = 10). b A western blot of Bat3-Myc, Tim-3–HA, and Lnc-Tim3 (wild type or Δ226-277) overexpressed in HEK 293 T cells and coimmunoprecipitated to confirm the specificity of the Bat3–Tim-3 interaction in mammalian cells. IP, immunoprecipitation; IB, immunoblot. Representative of three experiments. c A western blot of pLck (Tyr394 and Tyr505) expression from Jurkat T cells that were transfected with indicated lentiviral particles (Tim-3–HA, Lnc-Tim3 wild type or Δ226-277) and anti-TCR/anti-CD28 beads. Representative of three experiments. (d) Lnc-Tim3^highTim-3⁺CD8 T cells isolated from tumor-infiltrating lymphocytes (TILs) of HCC patients with Lnc-Tim3 high expression were transfected with indicated lentiviral particles (Lnc-Tim3 scramble or shRNA) and treated with anti-TCR/anti-CD28 beads. The expression of pLck (Tyr394 and Tyr505) was assessed by western blot. Representative of three experiments. e Jurkat T cells were transfected with indicated lentiviral particles (Tim-3–HA, Lnc-Tim3 wild type or Δ226-277) and treated with anti-TCR/anti-CD28 beads and PMA-ionomycin, followed by coimmunoprecipitated to confirm the specificity of the Bat3-pLck (Tyr394 and Tyr505) interaction in cells. Representative of three experiments. f Jurkat T cells were transfected with indicated lentiviral particles (Tim-3–HA, Lnc-Tim3 wild type or Δ226-277). Then, Jurkat T cells were also transfected with NFAT1 or AP-1 luciferase reporter. NFAT1 or AP-1 luciferase activity was determined after a 6-h stimulation (anti-TCR/anti-CD28 beads and PMA-ionomycin). Data are presented as means ± S.E.M. (*P < 0.05)