Mechanisms supporting APC/C<sup>Cdh1</sup> activity under ER stress conditions.

Abstract

<p>(A) HeLa cells were treated with DMSO, 0.5 µg/ml of TM alone, or 0.5 µg/ml of TM plus 5 µM of MG-132 for 16 h. Immunoprecipitates of endogenous Cdc27 were immunoblotted for endogenous Cdh1. Immunoprecipitation using IgG served as negative control. ** indicates Cdh1-specific top band; *indicates non-specific bottom band. (B) HeLa cells were treated with DMSO or 1 µg/ml of TM for 16 h. CDK2 or CDK1 antibodies were used to immunoprecipitate endogenous CDK2 or CDK1 complexes, respectively. Immunoprecipitates were then used in <i>in vitro</i> kinase assays using histone H1 as substrate. <i>Left</i>, autoradiography of <sup>32</sup>P-histone H1 phosphorylated by CDK2 complexes. Coomassie stains input of histone H1 in the reactions. Intensity of the autoradioactive bands were quantified by Image J, normalized to histone H1 input, and presented as arbitrary units (A.U.). Immunoblot shows comparable levels of endogenous CDK2 and the indicated proteins before and after TM treatment. <i>Right</i>, autoradiography of <sup>32</sup>P-histone H1 phosphorylated by CDK1 complexes, analyzed as indicated for CDK2. (C) <i>Left</i>, total cell lysates from HeLa cells treated with DMSO or 1 µg/ml of TM for 16 h were immunoblotted for the indicated endogenous proteins. <i>Right</i>, quantification of endogenous Emi1 mRNA levels in HeLa cells treated with DMSO or 1 µg/ml of TM for 16 h, measured by SYBR-green qRT-PCR.</p

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