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. Author manuscript; available in PMC: 2015 Oct 2.
Published in final edited form as: Brain Res. 2014 Aug 21;1583:230–236. doi: 10.1016/j.brainres.2014.08.018

Figure 4.

Figure 4

Unmasking of NOTCH3 N-terminal epitopes in recombinant NOTCH3 protein. Recombinant fusions of NOTCH3 ectodomain to mouse Fc were purified to homogeneity from 293 cell lines. Fifty ng of wild type NOTCH3 (WT), R90C NOTCH3 (R90C), or mouse Fc (C) were treated with chemicals indicated for 60 minutes at 37°C (or at 65°C without chemicals) and then spotted on nitrocellulose. 5210 or 2079 were used to detect N-terminal NOTCH3 epitopes. Untreated protein (UN) was applied on the left-most column. Conditions included 10 mM dithiothreitol (DTT), heat, 1% SDS, 6M urea, 6M guanidinium HCl (Guan), 0.3% acrylamide (Acry), and 0.5M hydrochloric acid (HCl). To verify presence of proteins, blots were also probed against Fc (bottom panel).