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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. 1 — a Strategy for identifying cFLIP_L-interacting E3 ubiquitin ligase by AlphaScreening. When biotinylated E3 ligase interacts with FLAG-tagged cFLIP_L, singlet oxygen generated from donor beads is transferred to acceptor beads, resulting in emission. b The top seven E3 ligases exhibiting high binding to cFLIP_L. Relative ratios were calculated by dividing the relative intensity of binding to cFLIP_L for each E3 ligase by the relative intensity of binding to dihydrofolate reductase. Results are the average ratios of two independent experiments. c HEK293 cells were transfected with the indicated E3 ligases along with FLAG-cFLIP_L and HA-Ub. After immunoprecipitation with anti-FLAG antibody, ubiquitylation and total amounts of immunoprecipitated cFLIP_L were analysed by immunoblotting with the indicated antibodies. Expression of transfected proteins and ubiquitylated proteins were verified by the indicated antibodies using cell lysates. d HEK293 cells were transfected and analysed as in (c). Because transfection of caspase 8 rapidly induced cell death, we used a protease-inactive mutant of caspase 8 (C8C/S). e HEK293 cells were transfected with the indicated expression vectors. Cell lysates were immunoprecipitated with control Ig (C) or anti-Myc (M) antibodies, and co-immunoprecipitated and immunoprecipitated proteins were analysed by immunoblotting with anti-FLAG and anti-Myc antibodies, respectively. Protein expression was verified in cell lysates using the indicated antibodies. f Endogenous cFLIP_L interacts with MIB2. HeLa cells were immunoprecipitated with control Ig (C) or anti-cFLIP antibody. Co-immunoprecipitated MIB2 was analysed by immunoblotting with anti-MIB2 antibody. Immunoprecipitated cFLIP_L was analysed by anti-cFLIP 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. Results are representative of two independent experiments (c–f).