Supplemental Data SUMO-2/3 Modification and Binding Regulate the Association of CENP-E with Kinetochores and Progression through Mitosis

Figure S1. Characterization of SUMO-1 and SUMO-2/3 specific monoclonal antibodies. A. Immunoblot analysis of recombinant SUMO-1, SUMO-2, and SUMO-3 proteins with SUMO-2/3 and SUMO-1 specific mAbs 8A2 and 21C7. 8A2 recognizes recombinant SUMO-2 and SUMO-3, but not SUMO-1. In contrast, 21C7 recognizes recombinant SUMO-1, but not SUMO-2 or SUMO-3. B. Comparison of the subcellular localizations of SUMO-2/3 and SUMO-1 in interphase cells. HeLa cells were fixed with formaldehyde, permeabilized with acetone, and analyzed by immunofluorescence confocal microscopy. Cells were double labeled with PML, SUMO-2/3, or SUMO-1 specific mAbs. SUMO-1 and SUMO-2/3 show similar localization patterns, both being detected throughout the nucleoplasm and concentrated in bright nuclear foci corresponding to PML nuclear bodies. Notably, however, only SUMO-1 is detected at the nuclear envelope and in nucleoli. Bar equals 5 µm. C. Comparison of SUMO-1 and SUMO-2/3 modified protein profiles. HeLa whole cell lysates were analyzed by immunoblotting with SUMO-1 or SUMO-2/3 specific antibodies. SUMO-2/3 are associated with high molecular mass conjugates that appear as a continuous smear beginning at ~150 kDa and extending to the top of the gel. SUMO-1 modified proteins also appear as a high molecular mass smear extending from ~80 kDa to the top of the gel. In general, SUMO-1 modified proteins are less biased toward very high molecular mass conjugates. SUMO-1 specific antibodies also recognize a