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. 2017 Nov 21;8:1638. doi: 10.1038/s41467-017-01807-7

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© The Author(s) 2017

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 — HSF1, PARP13, and PARP1 form a ternary complex. a HEK293 cells were transfected with HA-tagged PARP proteins, and subjected to HSF1 immunoprecipitation and immunoblotting. Asterisks indicate the full-length products from the transfected PARP constructs. b hPARP13-His purified from bacteria was pulled down with purified GST, GST-hPARP1, GST-hHSF1, GST-hHSF2, or GST-hHSF4, and subjected to immunoblotting. Input of PARP13-His was also shown at the bottom. Asterisks indicate full-length GST-fusion proteins. c Endogenous immunoprecipitation in nuclear fractions of HeLa cells, in which PARP1 or PARP13 was knocked down by infection with adenovirus expressing the corresponding shRNA, or scrambled RNA (SCR) as control. Input: PARP1, 1%; PARP13, 5%; HSF1, 1%. Immunoprecipitate (IP), 100%. Percentages of co-precipitated PARPs are shown. Nuclear (N) and cytoplasmic (C) fractions were blotted using an antibody for nuclear (SP1) or cytoplasmic (HSP90) protein. d The DNA-binding domain of HSF1 interacts with PARP13. GST-pull-down from mixtures of purified hPARP13-His and GST-fused hHSF1 mutants was performed, and proteins were subjected to immunoblotting. DBD DNA-binding domain; HR hydrophobic heptad repeat; DHR downstream of HR-C. e Alignment of the amino-acid sequences of the DNA-binding domains in hHSF1, hHSF2, and hHSF4. Thirteen residues in HSF1 differing among three sequences were substituted with alanine (black dots) or glutamic acid (white dots). Predicted secondary structure including four α-helices (boxes H1 to H3 and a C-terminal α-helix C-H), four β-sheets (arrows β1 to β4), and a wing motif is shown. f HEK293 cells were transfected with wild-type and mutated hHSF1-HA, in which Thr20 or Ala33 was replaced with a corresponding amino acid from hHSF2 or hHSF4. Extracts of these cells were subjected to HA immunoprecipitation and immunoblotting. g HEK293 cells were transfected with wild-type and mutated HA-hPARP13 (ΔZ, ΔWWE, and ΔZ-ΔWWE). Extracts of these cells were subjected to HA immunoprecipitation and immunoblotting