Fig. 5

Analysis of the interaction between CD44-ICD and RUNX2. a. Equal amounts of nuclear lysates of PC3/PBS (lane 1 and 3) and PC3/DAPT (lane 2) were immunoprecipitated with CD44-ICD antibody (lane 1 and 2) and subjected to immunoblotting (IB) analyses with an antibody to RUNX2 (lane 1–3). b. Immunoblotting analysis of the nuclear lysates from PC3 cells treated with PBS or DAPT (panel B) with a nucleoporin antibody demonstrates an equal amount of nuclear proteins were used for immunoprecipitation analysis shown in Fig. 5a. c-e. PC3 cells (lane 1) and PC3/RUNX2 overexpressing cells (lane 2) were immunoblotted with an HA- (C), RUNX2 and GAPDH (E) antibody. f. Equal amounts of PC3 (lane 1 and 3) and PC3/RUNX2 (lane 2) cells were immunoprecipitated with an antibody to RUNX2 and subjected to IB analyses with a CD44-ICD antibody. One asterisk (*) represents the 20 kDa CD44 extracellular truncation fragment (CD44-EXT). g-h: Analysis of the localization of CD44 (green), RUNX2 (red), and DAPI (a nuclear counterstain; blue) in PC3 cells. g. Confocal microscopy shows overlay stainings for CD44/DAPI/RUNX2 (green/blue/red), RUNX2/DAPI (red/blue), and CD44/DAPI (green/blue) in PC3 cells. Scale bar-100 μm. h. The rectangle in panel G defines the area of the image which is magnified in panel H. Arrows point to regions of colocalization (yellow) of CD44 fragment “CD44-ICD” (green) and RUNX2 (red); wavy arrows (H; CD44 panel) point to areas where CD44-ICD is localized in the nucleus and colocalized with RUNX2 in the overlay panel. Scale bar-10 μm. These results represent one of the three experiments performed with similar results