Fig. 6: CREPT is associated with Aurora B.

a A mass spectrometry analysis of proteins associated with CREPT. A mass spectrometry analysis was performed to identify proteins in the complex precipitated by an antibody against CREPT (3E10). IgG was used as a control. Precipitants were analyzed by an SDS-PAGE and stained with Coomassie blue. Bands were cut out and identified by a mass spectrometry analysis. b Aurora B and CREPT are coordinately expressed at the G2/M phase. Cells were synchronized by double thymidine blocks and released into complete medium to allow cell entry into different phases. Samples were collected at the indicated time points and mitotic markers were analyzed by western blots. c The protein levels of CREPT and Aurora B are both increased in the G2/M stage. Nocodazole was added into medium at indicated time point and the cells were harvested at the same time. d CREPT specifically interacts with Aurora B in vitro. Flag-CREPT and HA-Aurora B were transfected into HEK293T cells and harvested for the precipitation after 24–36 h. Immunoprecipitation (IP) was performed using an antibody against Flag or HA. The complex was examined by a western blot using an antibody against HA or Flag. e CREPT and Aurora B physically interact in vitro. A GST pull-down assay was performed with purified GST and GST-CREPT proteins. HA-Aurora B was overexpressed in HEK293T cells. f Endogenous CREPT interacts with Aurora B in vivo. An IP experiment was performed with an anti-CREPT antibody. Experiments were performed in MGC803 cells and mouse embryonic stem cells (mES). g The interaction of Aurora B and CREPT occurs at the G2/M phase. The cells were synchronized by DTB and IP was performed with anti-CREPT antibody at different phases during the cell cycle. h CREPT interacts with Aurora B through the RPR domain. HA-Aurora B was co-expressed in HEK293T cells with Myc-tagged full length and two deletions of CREPT. Cell lysates were immunoprecipitated using an anti-Myc or anti-HA antibody