Figure 6.

The WC629 inhibitory antibody bridges the LNR and HD domains and retards deuteration of the S2 region. (A) Ribbon diagram of the Notch1 NRR, colored according to the effect of antibody binding. Regions exhibiting slower deuteration in the presence of the antibody (with an absolute difference in mass of at least 0.5 Da) are colored blue, and regions with no detectable change in the deuteration rate (an absolute difference in mass of less than 0.5 Da) are green. Gray regions correspond to segments for which no HX MS data were obtained. (B) Epitope fine mapping. The indicated wild-type and mutated Notch1 NRR minireceptors, which are divided at S1 into two subunits during maturation, were tagged with FLAG and HA epitopes at their N- and C-termini, respectively, and complexes were precipitated with an anti-HA antibody. The top panel shows co-precipitation of the FLAG- and HA-tagged subunits for all mutants tested, verifying S1 cleavage and stable subunit association. The bottom panel shows co-immunoprecipitation of the WC629 inhibitory antibody with the minireceptor. The bound WC629 antibody was detected with a goat anti-human-HRP antibody conjugate. BCL: BC-loop; S2L: S2 loop; SWN2: Notch2 sequence swap; CM: conditioned media; B: beads. The sequences of the mutations tested are listed in Supplementary Figure 5. (C) Point mutagenesis of N1714 and K1718 of the S2 loop. Left panel shows co-precipitation of the FLAG- and HA-tagged subunits and right panel shows co-immunoprecipitation of the WC629 inhibitory antibody with the minireceptor. Co-precipitation experiments were carried out as above in panel (B). See also Figures S2, S3 and S4, and Table S1..