Fig. 10. Cytoplasmic Alfy-positive structures partially colocalize with ubiquitin-containing profiles and are induced by a proteasome inhibitor. HeLa cells were treated with 50 µM PSI or left alone for 5 hours and then further chased for 18 hours without PSI in the medium. Cells were then double labelled against Alfy (red) and ubiquitin (antibody against mono- and polyubiquitin, FK2; green). In both control cells (A,B) and treated cells (C,D), we observed an apparent colocalization between Alfy and ubiquitin. The number of labelled structures had dramatically increased in the PSI-treated cells (C,D). A closer observation of Alfy-positive structures at high magnification revealed that the markers did not completely colocalize. It was especially evident that some Alfy labelling seemed to extend beyond the ubiquitin-labelled area (E, arrowheads).