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. 2016 Jun 21;26(8):914–934. doi: 10.1038/cr.2016.78

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Copyright © 2016 The Author(s)

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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Plk3 interacts with FADD and DISC formation is required for Plk3 activation. (A) HeLa cells were treated with CD95L and CHX for the indicated time periods. Endogenous FADD was immunoprecipitated with rabbit anti-FADD and immunoblotted against Plk3 and FADD. (B) Jurkat cells were treated with CD95L and CHX for the indicated time periods. Cytoplasmic and membrane fractions were separated using hypotonic buffer. Endogenous FADD was immunoprecipitated with rabbit anti-FADD and immunoblotted for Plk3 and FADD. (C) The potential interaction between Plk3 and FADD was monitored via in situ PLA. HeLa cells were treated with CD95L and CHX for the indicated time periods and labeled with the anti-Plk3 (rabbit) and anti-FADD (mouse) antibodies. Single antibody staining (Plk3 or FADD) was used as a control. Scale bar: 10 μm. The quantification is shown on the right. Each bar represents the mean value ± SD (n = 3). The differences between single and double antibody staining were statistically significant by Student's t-test (^*P ≤ 0.05). (D) HeLa cells were transfected with FADD siRNA (siFADD) or a control siRNA (siC) for 48 h followed by the treatment with CD95L and CHX for the indicated time periods. The lysates were immunoblotted against Plk3, FADD, caspase-8, cleaved caspase-8, pT273 caspase-8 and GAPDH. (E) HeLa cells were transfected with FADD siRNA (siFADD) or a control siRNA (siC). Cells were treated with CD95L and CHX for the indicated time periods. The lysates were immunoblotted against FADD (left panel). Immunoprecipitated endogenous Plk3 was incubated with recombinant GST-fused procaspase-8 for an in vitro kinase assay. Samples were immunoblotted against pT273 caspase-8, GST and Plk3 (right panel). (F) HeLa cells were transfected with CD95 siRNA (siCD95) or a control siRNA (siC) and then treated with CD95L and CHX for the indicated time periods. Lysates were immunoblotted against Plk3 and CD95 (upper panel). Immunoprecipitated Plk3 was incubated with GST-fused procaspase-8 for an in vitro kinase assay. Samples were immunoblotted against pT273 caspase-8, GST and Plk3 (lower panel). (G) HeLa Casp8-WT and Casp8-KO cells were treated with CD95L and CHX for the indicated time periods. Lysates were immunoblotted against Plk3, caspase-8, cleaved caspase-8, pT273 caspase-8 and vinculin (upper panel). Immunoprecipitated Plk3 was incubated with GST-fused procaspase-8 for an in vitro kinase assay. Samples were immunoblotted against pT273 caspase-8, GST and Plk3 (lower panel).