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. 2018 Jul 18;9:2812. doi: 10.1038/s41467-018-05097-5

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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. 6 — Tbkbp1 mediates IL-15-stimulated autophagy in NKT cells. a–d IB analysis of LC3 modification and p62 degradation (a, c) and Ulk1 phosphorylation (b, d) in whole-cell lysates of IL-15-stimulated WT or Tbkbp1-KO thymic NKT cells (a, b) or NKT hybridoma cells transduced with a non-silencing control shRNA (shCtrl) or two different Tbkbp1-specific shRNAs that were stimulated with IL-15 (d) or IL-15 together with DMSO or bafilomycin A (c). e Confocal microscopic analysis of LC3 puncta, DAPI (nuclear staining), and merged picture of untreated (NT) or IL-15-stimulated control or Tbkbp1-knockdown NKT hybridoma cells. Data are presented as representative images (left) and summary graph of quantified LC3 puncta (right). Scale bar, 5 μm. f Flow cytometric analysis of intracellular Bcl2 expression level in WT or Tbkbp1-KO thymic NKT cells cultured for 2 days with medium control, IL-15, or IL-15 plus the autophagy inhibitor 3MA. g Summary graph of flow cytometric analysis of AnnexinV⁺ apoptotic cells in enriched thymic NKT cells that were either untreated (NT) or cultured for 1 day with the indicated agents. h, i Co-IP analysis of Tbkbp1-FIP200 (h) or Tbkbp1-Ulk1(i) interaction (upper) and direct IB assays (lower) using lysates of HEK293 cells transfected with the indicated expression vectors. j IB analysis of the indicated proteins in IP samples of control IgG, anti-FIP200, or anti-Ulk1 or direct lysates of NKT hybridoma cells that were stimulated as indicated. k Schematic of Tbkbp1 WT and mutants showing the coiled-coil domains (CCs), TBK-binding domain (TBD), proline-rich domain, and zinc fingers (ZF). l, m Co-IP analysis of Ulk1 binding with the indicated Tbkbp1 mutants (upper) and direct IB assays (lower) using HEK293 cells transfected with Myc-ULk1 along with Tbkbp1 truncation mutants (l) and point mutants (m). n IB analysis of the indicated phosphorylated (P-) or total proteins in whole-cell lysates of Tbkbp1-knockdown NKT hybridoma cells reconstituted with a vector control (Ctrl) or the indicated Tbkbp1 point mutants, stimulated as indicated. Data are representative of three or more independent experiments. **P < 0.01; ***P < 0.001. Two-way ANOVA (e), Student’s t-test (g)