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. 2022 Sep 26;50(18):10469–10486. doi: 10.1093/nar/gkac808

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — SIAH2 interacts with CtIP. (A) HEK293T cells were transfected with Flag-SIAH2 along with or without HA-CtIP. Total cell lysates were immunoprecipitated with either control IgG or anti-Flag antibody and then detected with the indicated antibodies. (B) HeLa cells were lysed either 30 min after ionizing radiation (IR) or 3 h after hydroxyurea (HU) treatment and subjected to immunoprecipitation followed by immunoblotting with the indicated antibodies. (C) Interaction between Flag-SIAH2 and endogenous CtIP in either G1 or S/G2 phase of the cell cycle. Hela cells transfected with Flag-mock or Flag-SIAH2 were synchronized in G1 and S/G2, cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotting was performed with the indicated antibodies. (D) Schematic representation of the mutant CtIP constructs used in this study (E) HEK293T cells were cotransfected with each of the indicated HA-CtIP constructs and Flag-SIAH2 and subjected to immunoprecipitation and immunoblotting with the indicated antibodies. Asterisks indicate nonspecific bands. (F) Schematic representation of SIAH2 constructs used in this study. (G) Cellular lysates from HEK293T cells cotransfected with the indicated Flag-SIAH2 constructs and HA-CtIP, immunoprecipitated with anti-HA antibody and then immunoblotted with indicated antibodies. Asterisks indicate nonspecific bands. (H) Purified GST-SIAH2 WT, GST-SIAH2 ΔSBD, and GST-SIAH2 SBD expressed in bacteria were incubated with lysates from HeLa cells, and CtIP that coprecipitated with each GST-SIAH2 construct was analyzed by immunoblotting using the CtIP antibody. GST immunoblots show input GST-SIAH2 constructs. Asterisks indicate GST-SIAH2. (I) Purified GST-CtIP (700–799) expressed in bacteria was incubated with lysates from HeLa cells, and SIAH2 that coprecipitated with GST-CtIP (700–799) fragment was analyzed by immunoblotting using the SIAH2 and GST antibodies. GST immunoblots show input GST-CtIP constructs.