Figure 6: CD24 interacts with NPM during mitosis.

(a,b) Dynamic association between CD24 and NPM in DU145 cells. (a) Merged images displaying DNA (stained with DAPI, blue), NPM (green) and CD24 (red). Cells at prophase, metaphase and anaphase were identified by their chromatin distribution. The representative image was obtained from a Meta laser-scanning confocal microscope. (b) Overlay between CD24 (red) and NPM (green) signals of cells at the three indicated phases during mitosis. The intensity (y axis) of signals was simultaneously obtained from the laser-scanning cross-section with the major diameter and overlap of the red and the green signals was analysed using LSM image browser. (c) Reciprocal co-precipitation between CD24 and NPM in DU145 cells. The cells lysates were prepared after 2 h of release from nocodazale arrest. The cell lysates were precipitated with antibodies specific for either NPM or CD24 or IgG control. The precipitates were probed by western blot with indicated antibodies. (d) Localizing the CD24-binding ___domain in NPM to the regions of AA196-294 based on co-IP of Myc-tagged NPM fragments and HA-tagged CD24 in HA-CD24 and NPM-Myc co-transfected HEK293 cells. The association was confirmed by western blot with anti-HA and anti-Myc mAbs. All images have been reproduced three times. DAPI, 4',6-diamidino-2-phenylindole. Co-IP, co-immunoprecipitation; IgLC, immunoglobulin light chain; IP, immunoprecipitation; IB, immunoblot.