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. 2018 Jul 9;9:2372. doi: 10.1038/s41467-018-04590-1

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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. 3 — The intracellular domain of FAT1 interacts with and activates the Hippo kinase signalome. a Representative western blots against Hippo pathway components in lysates of exponentially growing HEK293 CD4ext and CD4-FAT1-TM/ICD stable cells. Control, parental HEK293 cell line. b Analysis of YAP1 phosphorylation after transient transfection with CD4 control or CD4-FAT1-TM/ICD chimera in WT or the corresponding CRISPR/Cas9 sgRNA engineered knockout HEK293 cells as indicated. HA-YAP1 immunoprecipitates were analyzed by phos-tag phosphorylation affinity shift electrophoresis and YAP1 western blotting. Retarded (phosphorylated) YAP1 is indicated by arrowheads. A representative blot is shown. c Analysis of YAP1 phosphorylation after transient cotransfection with Flag-YAP1 and CD4 control or CD4-FAT1-TM/ICD chimera in HEK293 cells pretreated with control (C) or TAOK1/2/3 siRNA (TAOKs) as indicated. Flag-YAP1 immunoprecipitates were analyzed by phos-tag electrophoresis and YAP1 western blotting. Retarded (phosphorylated) YAP1 is indicated by arrowheads. A representative blot is shown. d On the left, a scheme depicting the GST fusion proteins indicating the approximate location of functional motifs present in FAT1 and LATS1 and the subsequent GST-pulldown assay. On the right, representative western blots of pulldown experiments using GST fusion proteins and HEK293 total cell lysates. e Summary of mutant FAT1 ICD constructs (PPXY and PDZ binding site) and the depicted deletions and their ability to bind MST1 as assessed by pulldown assay. f siRNA-mediated knockdown on HEK293 of the different components of the Hippo signaling pathway and subsequent GST-FAT1-ICD pulldown on whole cells lysates. g Endogenous FAT1 immunoprecipitation by a monoclonal antibody recognizing is extracellular region. Exponentially growing HEK293 were transfected with FAT1 and FAT2 siRNAs for 48 h and then treated for 2 h at 4 °C with DMSO (−) or the reversible crosslinker DSP (+) prior to cell lysis and immunoprecipitation with anti-FAT1. Representative western blots are shown