Fig. 1

Mutant Ubiquilin-2 overexpression results in punctate intracellular accumulations in primary mixed neuroglia cultures. a. Cells transduced with AAV-UBQLN2(WT) show ubiquilin-2 immunoreactivity (green) diffusely present in the cytoplasm and cell processes with few small punctate accumulations. In contrast, UBQLN2 mutants (P497S, P497H, and P506T) result in large intracellular ubiquilin-2 accumulations both in neuronal soma and processes (red, labelled with MAP2). Ubiquilin-2 accumulations in processes have a “bead on a string”-like appearance particularly for P497H and P506T mutants. b. Some ubiquilin-2 accumulations (green) are outside of neurons and colocalized with astrocytes in culture, labeled with GFAP-imunoreactivity (red). c. Western blot of TX-soluble and insoluble fractions show that all UBQLN2 mutants and not WT accumulate in the TX-insoluble/SDS fraction, suggesting formation insoluble aggregates. d. Graph of d2EGFP signal normalized to actin in HEK293 cells transfected with WT and mutant ubiquilin-2. Both P497S and P506T mutants show impaired proteasomal degradation of the d2EGFP reporter compared to the P497H mutant and WT ubiquilin-2. *p < 0.05, **p < 0.01