Comparative effects of cysteine mutation location on NOTCH3 gel mobility.A, schematic showing sites of cysteines (black triangles) in the first three EGF repeats of NOTCH3 in the Fc-NOTCH3(1–3) construct. For this experiment, each of the 18 cysteines shown in NOTCH3 were mutated to a serine residue (red circle), resulting in 18 independent mutant clones, which were transfected into cultured cells, whose conditioned medium was analyzed for Fc protein mobility. B, Western blots of Protein A-agarose–enriched Fc-NOTCH3(1–3) proteins separated under nonreducing conditions. Wildtype protein was compared with 18 different mutants labeled 1 to 18. C, quantification of the ratio of upper band signal to total signal is shown and is the result of at least three independent experiments (∗p < 0.05 compared with wildtype control).