Effect of ubiquitination and neddylation on APP processing in 293T cells. (A) Western blot showing expression and conjugation of HisG-tagged ubiquitin and HisG-tagged Nedd-8. Lysates from transfected cells were probed with an anti-HisG antibody. (B) Effect of ubiquitin and Nedd-8 on APP processing as determined by ELISA for Aβ, β-NTF, and α-NTF.

Effect of SUMO-3 and SUMO-3(G93A) on APP and BACE expression. (A) Pulse–chase study of the kinetics of APP metabolism in 293T cells cotransfected with equal amounts of APP and the plasmid indicated. Immature (I) and mature (M, M²) APP species are indicated. (B) Dose–effect of SUMO-3 and mutant on APP expression. Equal amounts of proteins from lysates of cells transfected with the indicated plasmids for 48 h were probed with an anti-C-terminal APP antibody. (C) Western blot showing dose–effect of SUMO-3 and mutant on BACE expression. Equal amounts of proteins from cells transfected with indicated plasmids for 48 h were probed with an anti-BACE antibody.

SUMO-3 in human brain and its effect on APP processing in a human neuronal cell line. (A) SUMO-3 immunoreactivity in brain. ND, nondemented. Scale bars represent 20 and 100 μm for high and low magnifications, respectively. (B) Effect of SUMO-3 on Aβ, β-NTF, and α-NTF generation in SK-N-MC cells as determined by ELISA. (C) Schematic diagram summarizing effects of sumoylation on APP amyloidogenesis. Polysumoylation negatively (−) regulates but monosumoylation or undersumoylation positively (+) regulates Aβ production. SUMO proteins are indicated by small white circles.

SUMO-3 sumoylation regulates APP processing in 293T cells. (A) Diagram showing APP processing and antibody epitopes used in ELISA: 266.1 and 3D6 for Aβ, 8E5 and AF20 or 192wt for β-NTF, and 8E5 and 2H3 for α-NTF. Drawing not to scale. (B) Dose–effect of SUMO-3 expression on Aβ, β-NTF, and α-NTF generation from cells cotransfected with APP and without or with increasing amounts of SUMO-3 plasmids. All transfections contained 160-ng APP plasmids. The amount of SUMO-3 plasmids used were 0, 40, 160, and 640 ng in transfections corresponding to the columns from left to right. Vector DNAs were supplemented to bring the total amount of DNA to 800 ng for each transfection. A similar dilution scheme was used in other figures as well. The results were expressed as relative to the amount of APP-processing products indicated in each panel to the control. The control was APP transfection alone. (C) Western blot showing dose–effect of SUMO-3 on α-NTF production. Equal amounts of growth media from cells transfected as indicated were probed with 8E5 antibody. (D) Western blot showing SUMO-3 expression and protein sumoylation. Equal amounts of protein from lysates of cells transfected as indicated above were probed with affinity-purified SUMO-3 antibody. Arrows indicate SUMO dimer and trimer. HMW Proteins, high molecular weight proteins. (E) Western blot showing expression and sumoylation/desumoylation of SUMO-3 and mutants. Cells were mock-transfected or transfected with APP and SUMO plasmids, and lysates were probed with anti-SUMO-3 antibody. (F) Effect of SUMO-3 and mutants on Aβ, β-NTF, and α-NTF generation from transfected cells as determined by ELISA. (G) Dose–effect of SUMO-3(11R) on Aβ, β-NTF, and α-NTF generation as determined by ELISA. Each of the above experiments was carried out more than three times with equivalent results as those presented. *, Significantly different from APP transfection alone (P < 0.005, ANOVA post hoc tests).