Fig. 1: Mutant NOTCH3 forms larger multimers compared to WT NOTCH3.
From: Oligomerization, trans-reduction, and instability of mutant NOTCH3 in inherited vascular dementia
On nonreducing SDS-PAGE gels, we observed higher-order multimerization (green line) of the R90C protein at various time points compared to WT protein, which only formed Fc dimers (pink * and orange <) (a). Comparison of R90C and WT multimer protein content to total Fc-NOTCH3 protein content from the media demonstrated the increased presence of R90C multimers in the media (b). R90C multimer content did not change with increased hours post-transfection (b). Center line represents the mean; upper and lower lines designate the standard deviation. Recombinant WT and mutant NOTCH3 protein were purified from stably transfected cell lines, as previously described9. All proteins were examined on nonreducing 4–20% SDS-PAGE gels (ThermoFisher). Protein laddering was observed for all mutants examined: R90C mutant, C49Y mutant, R75P mutant, and R141C mutant (c). Little to no laddering was observed for WT protein (c). The addition of ß-mercaptoethanol completely reduced multimers to the monomer level (d). Experiments were performed at least three times on distinct samples with similar results. Unprocessed gels can be found in Fig. S8.
