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Fig. 4 | Acta Neuropathologica Communications

Fig. 4

From: Composition of the Intranuclear Inclusions of Fragile X-associated Tremor/Ataxia Syndrome

Fig. 4

a FXTAS nuclear protein samples exhibit over ten times higher SUMO 2/3 protein levels compared to control nuclear protein samples. FXTAS nuclear protein samples exhibit a vibrant SUMO 2/3 smear indicating over 10 times higher levels of conjugated SUMO 2/3 over control nuclear protein samples after normalizing to total protein levels (unpaired student’s t-test (single group) with p-value <0.005). Whole tissue sample signals were extremely faint by comparison. No significant difference was seen between control and FXTAS samples for unconjugated SUMO 2/3. b SUMO 2/3 immunofluorescence reveals that SUMO 2/3 is present mainly as large, concentrated intranuclear aggregates in FXTAS brain cells, and FXTAS inclusions always colocalize with SUMO 2/3 aggregation. SUMO 2/3 forms large, intranuclear aggregates in FXTAS brain cells that colocalize with FXTAS inclusions. Immunofluorescence was performed using an Alexa 647 secondary antibody to minimize the effect of inclusion autofluorescence. 360nm+480nm images show inclusion autofluorescence. In FXTAS samples, SUMO 2/3 aggregates at 620nm are so vibrant that the signal intensity at 208.1ms is distorted, and the signal is bright even at an exposure of 10.1ms. No post-processing was done to alter brightness/contrast levels. Orange arrows denote FXTAS inclusions. When looking at clusters of nuclei (bottom panels), control brain cells show diffuse SUMO 2/3 staining throughout nuclei, but no aggregates, whereas FXTAS brain cells show large, circular aggregates in a large proportion of cells. c SUMO 2/3 aggregates are present in mouse high premutation CGG brain nuclei but are not present in wildtype brain nuclei. Intranuclear SUMO 2/3 aggregates are also seen in 11-month-old premutation CGG male mouse brain, but not in age-matched male wildtype mice. Aggregates were smaller and fewer than those seen in human FXTAS brain. Immunofluorescence was performed using an Alexa-647 secondary antibody. No postprocessing was done to alter brightness/contrast levels. Orange arrows denote SUMO 2/3 aggregates. d The SUMO 2/3 smears found in FXTAS nuclear protein samples are not present to the same degree in other neurodegenerative brain samples investigated. Western blot analysis was performed on nuclear lysates from human frontal cortex tissue obtained from a variety of neurodegenerative disorders. Due to the lack of availability of sufficient material, replicates were not possible for several samples. Where replicates were possible, error bars are shown. Scale bars = 10 μm

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