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. 2020 Apr 28;11(4):298. doi: 10.1038/s41419-020-2493-1

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© The Author(s) 2020

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. 2 — a, b Chk1 impacts on interaction between βTRCP1 and Cdh1 both in G1-S extracts and in vitro. a HeLa cells were treated with HU (2 mM) for 20 h to arrest them at the G1-S boundary and extracts were prepared. The G1-S extracts were treated with Chk1 inhibitor, CHIR-124 (500 nM) where indicated before incubating with GST-Cdh1. After phosphorylation, GST-Cdh1 was first bound by glutathione beads and then mixed with in vitro translated HA-βTRCP1 for an hour. Immunoblot analysis was carried out to detect the bound HA-βTRCP1 protein. Right panel, quantification of the band intensities. Co-precipiated HA-βTRCP1 band intensities were normalized to the respective GST-Cdh1 bands and then further normalized to vehicle control (-). Data are represented as mean ± SD, **p < 0.005 by ANOVA with Holm-Sidak post-test, n = 3. b Immunoblots showing that the in vitro phosphorylation of GST-Cdh1 by Chk1 promotes its binding with HA-βTRCP1. c, d Chk1 activity is crucial for the interaction of Cdh1 with βTRCP1 in vivo. c Immunoblot analysis of immunoprecipitates and whole-cell lysates derived from 293T cells transfected with HA-Cdh1 and Myc-Chk1 (where indicated). 30 h post-transfection, cells were treated with proteasome inhibitor MG132 (10 µM) for 5 h and then harvested to do immunoprecipitation to detect the interaction between HA-Cdh1 and endogenous βTRCP1 proteins. Right panel, quantification of the band intensities (n = 3). Immunoprecipiated HA-Cdh1 band intensities were normalized to the respective βTRCP1 IP bands and then further normalized to Vector control (-). The dashed line and asterisk represents the removal of an intervening lane btween these two samples. d Immunoblot analysis of immunoprecipitates and whole-cell lysates derived from 293T cells transfected with both HA-Cdh1. 30 h post-transfection, Cells were treated with hydroxyurea (HU), Chk1 inhibitor (where indicated), and the proteasome inhibitor MG132 (10 µM) for 5 h before harvesting to do IPs. Right panel, quantification of the band intensities (n = 3). Immunoprecipiated HA-Cdh1 band intensities were normalized to the respective βTRCP1 IP bands and then further normalized to Vehicle-treated control (-). Data are presented as mean and SEM. Significance in panel (a) was determined with a one-way ANOVA and significance in panels (c) and (d) was determined by a one-tailed unpaired T-test (*p < 0.05 and **p < 0.005).