Figure 4.

Mutational analysis of Notch digestion by furin in vitro. (A) Schematic map of the recombinant substrate: murine Notch1 amino acids coordinates are indicated below the map. The main processing site (RQRR) and the two potential secondary sites are shown above the map, and the positions where processing by furin takes place are shown below. (B) The wild-type (WT, lanes 2 and 3) or the mutated (RQRR → AAAA; Mut, lanes 4 and 5) recombinant substrates were either mock-digested (lanes 3 and 4) or digested with purified recombinant BCRD-furin (lanes 2 and 5), and the products were analyzed on 15% Tricine gels followed by silver staining. The full-length substrate and N-terminal digestion products are indicated by dots on the left, and the C-terminal digestion products are indicated by arrows. The digestion product, purified on nickel-agarose, used for N-terminal sequencing is shown in lane 1. Molecular mass markers (kDa) are indicated on the right.