Figure 3.
EG5 is a UHRF1-interactive mitotic protein. (A) EG5 was identified as a component of UHRF1-interactive protein complexes by immunoprecipitation coupled with mass spectrometry. UHRF1-interacting protein complexes were immunoprecipitated with anti-Flag antibody in HEK293T cells expressing Flag-tagged UHRF1, and then were eluted with Flag peptide. The UHRF1-interacted protein complexes were separated by SDS-PAGE and a 125-kD electrophoretic band was subjected to mass spectrometry analysis. (B) HEK293T, DU145, and PC3 cells were transfected with plasmids expressing UHRF1-His or EG5-Flag, and the interaction between exogenous UHRF1 and EG5 proteins was validated by immunoprecipitation with antibodies against His or Flag, followed by immunoblotting. (C) The interaction between endogenous UHRF1 and EG5 proteins was validated by immunoprecipitation with antibodies against UHRF1 or EG5, followed by immunoblotting in HEK293T, DU145, and PC3 cells. (D) DU145 cells were synchronized in G1/S phase using double thymidine blocking and then released by culture media. The cells were synchronized in G2/M phases at 9 h after release. The interaction between endogenous UHRF1 and EG5 proteins was validated by immunoprecipitation with antibodies against UHRF1, followed by immunoblotting. (E and F) GST-tagged full-length UHRF1 and individual subdomains were constructed for mapping the EG5-binding region. Purified recombinant proteins of GST-tagged individual domains of UHRF1 were incubated with HEK293T cell lysates in vitro as indicated, followed by immunoblotting with anti-EG5 antibody (F). The lysate of HEK293T cells was used for a positive control. (G and H) GST-tagged full-length EG5 and individual subdomains were constructed for mapping the UHRF1-binding region. Purified recombinant proteins of GST-tagged individual domains of EG5 were incubated with HEK293T cell lysates in vitro as indicated, followed by immunoblotting with anti-UHRF1 antibody (H). The lysate of HEK293T cells was used for a positive control. Source data are available for this figure: SourceData F3.

EG5 is a UHRF1-interactive mitotic protein. (A) EG5 was identified as a component of UHRF1-interactive protein complexes by immunoprecipitation coupled with mass spectrometry. UHRF1-interacting protein complexes were immunoprecipitated with anti-Flag antibody in HEK293T cells expressing Flag-tagged UHRF1, and then were eluted with Flag peptide. The UHRF1-interacted protein complexes were separated by SDS-PAGE and a 125-kD electrophoretic band was subjected to mass spectrometry analysis. (B) HEK293T, DU145, and PC3 cells were transfected with plasmids expressing UHRF1-His or EG5-Flag, and the interaction between exogenous UHRF1 and EG5 proteins was validated by immunoprecipitation with antibodies against His or Flag, followed by immunoblotting. (C) The interaction between endogenous UHRF1 and EG5 proteins was validated by immunoprecipitation with antibodies against UHRF1 or EG5, followed by immunoblotting in HEK293T, DU145, and PC3 cells. (D) DU145 cells were synchronized in G1/S phase using double thymidine blocking and then released by culture media. The cells were synchronized in G2/M phases at 9 h after release. The interaction between endogenous UHRF1 and EG5 proteins was validated by immunoprecipitation with antibodies against UHRF1, followed by immunoblotting. (E and F) GST-tagged full-length UHRF1 and individual subdomains were constructed for mapping the EG5-binding region. Purified recombinant proteins of GST-tagged individual domains of UHRF1 were incubated with HEK293T cell lysates in vitro as indicated, followed by immunoblotting with anti-EG5 antibody (F). The lysate of HEK293T cells was used for a positive control. (G and H) GST-tagged full-length EG5 and individual subdomains were constructed for mapping the UHRF1-binding region. Purified recombinant proteins of GST-tagged individual domains of EG5 were incubated with HEK293T cell lysates in vitro as indicated, followed by immunoblotting with anti-UHRF1 antibody (H). The lysate of HEK293T cells was used for a positive control. Source data are available for this figure: SourceData F3.

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