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. 2021 Jan 19;4:80. doi: 10.1038/s42003-020-01603-y

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© The Author(s) 2021

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. 10 — a MIB2 KO HeLa/HA-MIB2 WT cells were untreated or treated with TNF (10 ng/ml) for the indicated times, and then lysates were immunoprecipitated with control (C) or anti-HA antibody (H). Co-immunoprecipitated proteins were analysed by immunoblotting with the indicated antibodies. Protein expression was verified by the indicated antibodies using cell lysates. b The RIPK1/MIB2 complex and cFLIP_L/MIB2 complex independently exist in the cells. HeLa cells were immunoprecipitated with control Ig (C) or anti-cFLIP antibody, and co-immunoprecipitated proteins were analysed by immunoblotting with the indicated antibody. The upper and lower arrowheads indicate modified and unmodified cFLIP_L, respectively. Asterisks indicate degraded bands of cFLIP_L. Protein expression was verified by the indicated antibodies using cell lysates. c MIB2 KO HeLa/HA-MIB2 WT or HA-MIB2 MT cells were untreated or treated with TNF (10 ng/ml) for the indicated times, and lysates were immunoprecipitated and analysed as in a. d MIB2 KO (KO), MIB2 KO HeLa/HA-MIB2 WT (KO WT), or HA-MIB2 MT (KO MT) cells were untreated or treated with TNF (10 ng/ml)/BV6 (0.1 μM) in the absence or presence of Nec-1s (20 μM) for the indicated times. Cell lysates were analysed by immunoblotting with the indicated antibodies. All results are representative of two or three independent experiments. e When WT cells are stimulated with TNF/IAP inhibitor, autophosphorylation of RIPK1 is prevented by MIB2, therefore RIPK1 cannot undergo complex IIb formation. Although caspase 8 and ubiquitylated cFLIP_L are recruited to the RIPK1/FADD complex, ubiquitylated cFLIP_L prevents a proper oligomer formation with caspase 8, thereby suppressing caspase 8 activation and apoptosis. In contrast, when MIB2 KO cells are stimulated with TNF/IAP inhibitor, autophosphorylation of RIPK1 is induced and phosphorylated RIPK1 subsequently forms the complex IIb. Recruited un-ubiquitylated cFLIP_L forms a heterodimer with caspase 8 but fails to block caspase 8 activation, thereby inducing apoptosis.