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. 2021 Jul 23;118(30):e2102502118. doi: 10.1073/pnas.2102502118

Fig. 3.

Fig. 3.

Notch1fe is flexible and the NRR dimerizes weakly. (A) Schematic representation of the interaction and biophysical experiments on regions reported in B–H. (B–D) Structural analysis of monomeric Notch1fe from SEC-SAXS, including the Dimensionless Kratky plot with crosshairs indicating the peak position for a globular protein (B), Guinier plot with a black line indicating the fit used to derive the Rg (C), and pair distance distribution function (D). (E) SPR equilibrium-binding plot of Notch1EGF33–NRR to Notch1EGF8–13. (F) SEC-MALS analysis of Notch1fe shows a monomeric and monodisperse sample (thick lines indicate the molecular weight, left axis). (Inset) Coomassie-stained SDS-PAGE of purified Notch1fe in reducing conditions. UV, ultraviolet. (G) Coomassie-stained SDS-PAGE of purified Notch1fe in nonreducing conditions. MW, molecular weight. (H) SEC-MALS analysis of Notch1NRR at three concentrations, determined at elution, shows a monomer–dimer equilibrium (thick lines indicate the molecular weight, left axis). (Inset) Coomassie-stained SDS-PAGE of purified Notch1NRR in reducing conditions, note that Notch1NRR is processed at the S1 cleavage site into two fragments of 8 and 27 kDa.