Journal: The Journal of Biological Chemistry

Article Title: Hotspot site microenvironment in the deubiquitinase OTUB1 drives its stability and aggregation

doi: 10.1016/j.jbc.2024.107315

Figure Lengend Snippet: Heterotypic aggregation: in-vitro coaggregation of OTUB1 and α-Synuclein. A and B , seeding effects of OTUB1 aggregates on the aggregation of α-Synuclein were studied by using ThT binding kinetics ( A ) and atomic force microscopy ( B ). OTUB1 seeds (5 μM) were added at 0 h to 300 μM of α-Synuclein and aggregation kinetics were performed using ThT dye binding assay. Aliquotes at different time intervals were collected to measure ThT fluorescence and for AFM sample preparation. The ThT data was plotted in GraphPad Prism and fitted using a non-linear fit sigmoidal model, and subjected to a two-way ANOVA (Dunnett’s multiple comparison test) to assess statistical significance. ∗∗∗∗ denotes a p -value of <0.0001. All the AFM samples were prepared on freshly cleaved mica surfaces, and imaging was performed in intermittent tapping mode using Bruker NanoWizard 3 (JPK instruments); data was processed using the JPK data processing tool (scale bar- 1 μm). C , cross-seeding assay by sonicated α-Synuclein pre-formed fibrils shows they can nucleate wild-type ( violet ) but not the mutants F133A (black) and V173K ( orange ) as observed from the light scattering assay.

Article Snippet: All the AFM samples were prepared on freshly cleaved mica surfaces, and imaging was performed in intermittent tapping mode using Bruker NanoWizard 3 (JPK instruments); data was processed using the JPK data processing tool (scale bar- 1 μm).

Techniques: In Vitro, Binding Assay, Microscopy, Fluorescence, Sample Prep, Comparison, Imaging, Cross Seeding Assay, Sonication, Scattering Assay