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. 2021 Dec 2;4:1330. doi: 10.1038/s42003-021-02853-0

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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 Scheme for Rapid immunoprecipitation Mass spectrometry of Endogenous protein (RIME) assays. Two antibodies anti- Flag (for K-Rta) and anti-RNAPII (POLR2A) were used to identify cellular proteins that exhibited increased interactions with POLR2A during KSHV reactivation. b STRING Interaction map. List of protein names among RNAPII-interacting proteins whose peptide counts were increased >1.5-fold in the presence of K-Rta with p-value <0.05 are plotted with STRING. c Co-immunoprecipitation. KSHV reactivation was induced by inducing exogenous K-Rta expression from a tetracycline-inducible promoter, and immunoprecipitation was performed with anti-K-Rta antibody. The indicated protein was probed for using the corresponding specific antibody. Total cell lysates were used as input control. d Gene ontology of recruited proteins via K-Rta. Protein names were subjected to GO analyses and the six highest protein functions are presented in the table. e Schematic diagram of K-Rta protein domains and intrinsically disordered protein (IDP) plot. Previously published K-Rta-function domains are indicated in the diagram. The transactivation domain, with which K-Rta interacts with the coactivator complex, is marked in magenta. The IDP score was calculated with a web-interface (https://iupred2a.elte.hu/) and plotted. The K-Rta transactivation domain is located at an intrinsically disordered region (IDR). f K-Rta and mutant transactivation domain sequence. Recombinant GST-protein sequences used for GST-pull down assays are shown. Altered amino acid sequences in the mutant are depicted in the bottom of the panel. g Purified GST-K-Rta deletion proteins and SWI/SNF components used for pull-down. Coomassie staining of SDS-PAGE gels are shown in the left panel. Molecular size marker is indicated left side of gel. h GST-pull down assays. The indicated purified SWI/SNF proteins were mixed and incubated with GST-K-Rta peptides. Immunoblotting with anti-Flag tag antibody was performed to probe interactions.