Fig. 1: The N-terminal region of ID2 including Ser-5 is necessary for the interaction between ID2 and core APC subunits. | Nature Communications

Fig. 1: The N-terminal region of ID2 including Ser-5 is necessary for the interaction between ID2 and core APC subunits.

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

Fig. 1

a GST-ID2 WT and deletion mutants of the N- and C-terminus were incubated with lysates from HeLa cells. Bound proteins were analyzed by western blot. Lower panel, Coomassie staining of GST-ID2 proteins used in the GST pull-down. b GST pull-down using GST-ID2 WT and N-terminus deletion mutants and HeLa cell lysates. Bound proteins were analyzed by western blot. Lower panel, Coomassie staining of GST-ID2 proteins used in the GST pull-down. c GST pull-down using GST-ID2 proteins and U-2 OS cell lysates followed by western blot. Arrowhead, specific band; asterisk, non-specific band. Lower panel, Coomassie staining of GST-ID2 proteins used in the GST pull-down. d In vivo interaction between ID family members expressed in HeLa cells and core APC subunits, CDH1 coactivator, and bHLH transcription factors (E47 or HEB). ID interacting proteins were detected by FLAG immunoprecipitation followed by western blot for endogenous proteins as indicated. e Interaction between ID2 phosphorylation mutants and D-Box mutant (DBM) expressed in HeLa cells and core APC subunits or CDH1 coactivator. ID2 interacting proteins were detected by FLAG immunoprecipitation followed by western blot for endogenous proteins as indicated. f FLAG-ID2-S5 phospho-mutants expressed in HeLa cells were immunoprecipitated and probed by western blot for the association with endogenous E47. g HeLa cells transduced with non-targeting or CDH1 siRNA were transfected with FLAG-ID2 WT or ID2-S5 phospho-mutants. Cellular lysates were used in FLAG immunoprecipitation followed by western blot for core APC subunits and CDH1 (left panel). Right panel, whole cellular lysates. Molecular weight markers are indicated in kDa. Coomassie staining in a–c was performed on SDS-gels loaded with the same GST-fusion protein amounts used in the binding reactions. APC proteins and CDH1 are from the same blots in each panel; E47 and HEB in panel d are from two independent gels; FLAG is from an independent gel; Loading controls are from the same gel as HEB in d, FLAG in e, E47 in f, APC1/APC3/CDH1 in g. Experiments were repeated two times with similar results.

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