Fig. 2

Biochemical and biophysical characterization of recombinant MSI1 and MSI2 protein aggregates (a) Western blot images showing in vitro aggregation of recombinant MSI1 and MSI2 aggregates as revealed by immunoblotting with α-Oligomer antibody, F11G3. Generic α-MSI1 antibody detected recombinant MSI1 aggregates, as well as Aβ aggregates. Whereas, α-MSI2 antibody detected only recombinant MSI2 aggregates. (b) AFM images of MSI1 and MSI2 protein aggregates showed different morphologies. MSI1 aggregates appeared as pear-shaped oligomers, while MSI2 had mostly spheroid oligomers. Proto-fibrils formation was observed in both MSI1 and MSI2 oligomers (black arrows, black scale bar: 100 nm). (c) GST pull down assay of MSI and tau demonstrates their physical interaction. Western blot analysis of immunoprecipitated GST-MSI1 and GST-MSI2 (Lane 2: IP’d GST-MSI1-Tau; Lane 4: IP’d GST-MSI2-tau) with Tau 13 (Pan-tau antibody) revealed the presence of tau proteins in the immunoprecipitated material as well. The inputs from the two reaction mixtures are also shown here (Lane 1: Tau + MSI1 input; Lane 3: Tau + MSI2 input). (d) Representative Western blot images of brain homogenates from AD (N = 4) and age-matched control cases (N = 4) demonstrating a significant increase in the levels of both MSI1 and MSI2 proteins ((p = 0.035, *; p = 0.005, **). (e) Representative images of both soluble and insoluble fractions of AD (N = 3) and age-matched control (N = 3) brain tissues obtained from filter trap assay. Soluble fractions from all the cases studied demonstrate increased levels of tau protein upon probing with Tau 13 antibody when compared to their insoluble fractions. Similarly, both MSI1 and MSI2 protein levels are increased in the soluble fractions compared to the insoluble fractions (p = 0.0279, *; p = 0.0045, **)