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. 2018 Aug 28;9(9):860. doi: 10.1038/s41419-018-0921-2

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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. 2 — a Cells were transfected with Ctrl siRNA, IKKβ, or IKKα siRNA for 48 h, serum-starved overnight, and then treated with TNFα for 1 h. Treated cells were collected for western blot analysis of the expression of the protein shown in the figure. β-Actin was used as a protein loading control. p-S6K1^T389 means the phosphorylation status of S6K1. b Cells were transfected with HA-Ctrl, HA-ARD1 WT, or HA-ARD1 mutants for 48 h, serum-starved overnight, and treated with TNFα for 1 h. Treated cells were collected for western blot analysis of the expression of the protein shown in the figure. c Cells were cotransfected with FLAG-IKKβ, HA-Ctrl, HA-ARD1 WT, or HA-ARD1 mutants for 48 h, serum-starved overnight, and treated with TNFα for 1 h. Treated cells were collected for the detection of protein expression. d Lysates of HEK293T cells cotransfected with FLAG-IKKβ, Myc-ARD1, and HA-S6K1 were analyzed with antibodies directed against the HA tag by coimmunoprecipitation and immunoblotting by p-S6K1^T389 and HA. e HEK293T cells were cotransfected with FLAG-IKKβ, Myc-ARD1, and HA-4EBP1, and then the cells were lysed for coimmunoprecipitation assay as described in d. f HEK293T cells were cotransfected with FLAG-IKKβ, Myc-ARD1, and HA-TSC2 and then treated as described in d. g MCF-7 cells were serum-starved overnight and treated with TNFα for 1 h. Cell lysates were immunoprecipitated with specific antibodies to ARD1 and TSC2 to identify the association of endogenous proteins. h Indicated cells were transfected with HA-ARD1 WT or HA-ARD1 S209A, serum-starved overnight, and then treated with TNFα for 1 h. Cell lysates were collected for the detection of the change of p-S6K1^T389. i Treated cells as described in h were collected for the detection of cell viability and proliferation. Graphs showing results of quantitative analyses (n = 3, mean ± S.D. *P < 0.05)