Fig. 6: The APC substrates SKP2 and Cyclin B exhibit a phosphorylation-dependent mechanism for the interaction with core APC during cell cycle progression. | Nature Communications

Fig. 6: The APC substrates SKP2 and Cyclin B exhibit a phosphorylation-dependent mechanism for the interaction with core APC during cell cycle progression.

From: Regulated interaction of ID2 with the anaphase-promoting complex links progression through mitosis with reactivation of cell-type-specific transcription

Fig. 6

a Western blot of HeLa cells synchronized by double thymidine block using phospho-specific antibodies. b Western blot of immunoprecipitates from HeLa cells synchronized by double thymidine block and collected at serial times for immunoprecipitation using SKP2 (upper panel), Cyclin B (middle panel), or ID2 (lower panel) antibodies. E47 and HEB are controls for the immunoprecipitation of endogenous ID2. c Loss of CDH1-mediated regulation of SKP2-S64D stability. Western blot of HeLa cells expressing FLAG-SKP2 WT, S64A, or S64D in the presence or absence of CDH1. d Loss of CDH1-mediated regulation of Cyclin B-S126D stability. Western blot of HeLa cells expressing FLAG-Cyclin B WT, S126A, or S126D in the presence or absence of CDH1. e In vivo ubiquitylation of HeLa cells co-expressing FLAG-SKP2 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lyasates immunoprecipitated with FLAG antibody. WCL, whole cellular lysates. f In vivo ubiquitylation of HeLa cells co-expressing FLAG-Cyclin B1 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lysates immunoprecipitated with FLAG antibody. WCL whole cellular lysates. g Loss of interaction between the SKP2-S64D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-SKP2 WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). h Loss of interaction between the Cyclin B-S126D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-Cyclin B WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). Total and phosphorylated proteins are from independent gels; APC1, APC3, CDH1 are from the same gel in each panel; E47, HEB, pHH3, SKP2 and Cyclin B1 are from independent gels; HA and FLAG/Vinculin in panel e are from different gels; Loading controls are from the same gel as FLAG or HEB in panel a. Molecular weight markers are indicated in kDa. Experiments were repeated two times with similar results.

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