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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. 1 — a Inhibition of Chk1 increases the stability of endogenous Cdh1. Immunoblot analysis of whole-cell lysates derived from both HeLa and 293T cells treated with increasing doses of the Chk1 inhibitor (Chk1i), CHIR-124, for 5 h before harvesting. Lower panels, Cdh1 band intensities were normalized to actin and then further normalized to vehicle (n = 3). b Depletion of endogenous Chk1 leads to increased levels of endogenous Cdh1. Immunoblot analysis of whole-cell lysates derived from 293T cells transfected with the indicated siRNA (non-targeting siCTRL, −; siChk1, +). Lower panels, Cdh1 band intensities were normalized to actin and then further normalized to siCTRL transfected cells (n = 3). c Mitotic population does not contribute to the increased endogenous Cdh1 level after Chk1 inhibitor treatment. Immunoblot analysis of whole-cell lysates derived from HeLa cells after removal of the mitotic population via a mitotic shake-off post Chk1 inhibitor, CHIR-124 (500 nM) treatment for 5 h. Lower panel, Cdh1 band intensities were normalized to actin and then further normalized to vehicle-treated cells. d, e Inhibition of Chk1 increases the half-life of transfected Cdh1 in 293T cells. d Immunoblot analysis of whole-cell lysates derived from 293T cells, transfected with HA-Cdh1 and HA-βTRCP1 constructs. Cells were treated with both hydroxyurea (HU) and Chk1 inhibitor (Chk1i), CHIR-124 (500 nM), for another 4 h before addition of 50 µg/ml cycloheximide (CHX). At the indicated time points, whole-cell lysates were prepared for immunoblot analysis. e Quantification of the band intensities in (d). Cdh1 band intensities were normalized to actin and then further normalized to t = 0 controls. f, g Inhibition of Chk1 increases the half-life of endogenous Cdh1 in 293T cells. f. 293T cells were treated with both hydroxyurea (HU) and Chk1 inhibitor (Chk1i), CHIR-124 (500 nM), for another 4 h before addition of 50 µg/ml cycloheximide (CHX). At the indicated time points, whole-cell lysates were prepared for immunoblot analysis. g Quantification of the band intensities in (f) (n = 3). Mean and SEM are provided. Significance in panels (a), (b), and (g) was determined by a one-tailed unpaired T-test (*p < 0.05, **p < 0.005, and ***p < 0.0005).