Cul4B did not colocalize with nedd8. A. Cul4Bs were not neddylated in NT-2 cells. The top row shows a metaphase cell in which most Cul4Bs did not co-localize with nedd8. The middle role shows a cell from an old NT-2 that had a Cul4B-positive process where Cul4B was not neddylated. The bottom row shows G0-synchronized NT-2 cells in which nedd8 was induced by synchronization in 0.02% FBS for 48 h. These cells also had unneddylated Cul4Bs in processes. The space bars represent 5, 5, and 10 μm, respectively. B. Cul1 and nedd8 were detected in the nucleus in G0-synchronized NT2 cells. The space bar is 5 μm. C. Cul4Bs did not colocalize with nedd8 in vivo. Human brain hippocampus was stained with the anti-Cul4B and mouse anti-nedd8. The image was taken near SVZ. A tiny amount of punctate nedd8 (white arrows) was also observed and appeared to represent neddylated Cul4B-3. A huge amount of unneddylated Cul4B was found in some cells that had DNA less accessible by DAPI. The space bar is 10 μm. All images were obtained with a 40x objective.