Immunodetection of Aurora-A and Notch4, two targets of FBXW 7. (A) Detection of Aurora-A in human tumors by western blotting. The abundance of Aurora-A has been quantified in 5 randomly chosen G-IV and one representative G-II. The same blot has been hybridized with anti-actin antibody for loading control and normalization. Aurora-A is detected in 4 out of 5 G-IV with downregulation of FBXW 7. (B-K) Immunohistochemical detection of Aurora-A and Notch4 in human G-II (mid panel, bar = 10 μm) and in a primary invasive glioblastoma (lower panel, bar = 100 μm). Nuclei are labeled with DAPI (blue). In G-II, Aurora-A detection (red) is mostly sparse (B) and accumulates in only a few tumor areas (C). Notch4 (red) labels only α-SMA positive (green) vascular structures (D), but not Ki-67 positive (green) proliferating tumor cells (E). By contrast, in invasive primary G-IV (lower panel) of known histology and α-SMA labeling (F, I) discriminating vascular structures (v, vein ; a, arteriole), Aurora-A is massively detected in specific zones of G-IV, mostly around vessels (G-J). Notch4 is also detected in some tumor cells in addition to the endothelial lining of arterioles (H-K).