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. 2021 Sep 24;11:19022. doi: 10.1038/s41598-021-98218-y

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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PIGN forms a complex with SAC components. MAD1 and MAD2 were co-purified with PIGN in an HA-tag pull-down assay in PIGN null HEK293 cells ectopically expressing 3HA-tagged PIGN. (A,B) HA-tag IP in asynchronous cells indicated optimal 3HA-tagged PIGN expression and co-purification with MAD1 and MAD2 at the 48-h time point. (C) MAD1 and MAD2 were co-purified with PIGN in G2/M synchronized cells. (D) Co-immunoprecipitation (Co-IP) experiment showed that PIGN endogenously interacted with MAD1 and MAD2 during SAC activation via Taxol (60 ng/µl) or nocodazole (60–100 ng/µl) treatment of K562 cells for 12 h. (E) HA-tag pull-down assay with cells released from early S-phase into mitosis indicated PIGN co-purification with MAD1 and MPS1. Inputs represent 5–10% of total protein lysate. All western blot images were cut within the molecular weight ranges of the protein targets prior to hybridization. Images generated from these cut blots by scanning the developed films. Images were cropped and labeled using Adobe Photoshop CC 2017 (version 18). Where applicable, representative full images of the blots are presented in Figure 3 of the supplementary immunoblotting data.