Figure 1.

AXIN1 polymerization is a major contributor to the concentration-dependent DC stoichiometry

(A) Schematic representation of the DC. See Figure S1A for further details.

(B) DC variants containing either wild-type or polymerization-deficient (M3) AXIN1, affinity-purified via AXIN1-dStrepII, and analyzed using SDS-PAGE and Coomassie staining. FT, flowthrough.

(C) Quantification of total β-catenin recovered with AXIN1-dStrepII and its phosphorylation, normalized to recovered total β-catenin. Data are from immunoblot analyses of experiments as shown in (B). See Figure S1B for a representative set of immunoblots. Results are means from three independent experiments ± SEM. Statistical analyses were performed using an unpaired Student’s t test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

(D) SEC-MALS of the DC containing either wild-type or M3 AXIN1 at different input concentrations. Data points are mean weight average molecular weights (M_W) ± uncertainty (see STAR Methods). See Figure S1D for SDS-PAGE analysis of input samples and Figures S1E and S1F for SEC-MALS data and representative SDS-PAGE analyses of elution fractions.

(E) SEC-MALS of the DC containing either wild-type or M3 AXIN1 at a single input concentration. Light scattering intensities at 90° and calculated M_i values are plotted. Input A₂₈₀, M_W, and dispersities (Ð) over the peak areas with uncertainties are tabulated. Peak fractions were analyzed using SDS-PAGE and Sypro Ruby staining.

(F) Mass photometry of input and SEC-MALS elution fractions (indicated by colored dots in E).

(G) In silico modeling of DC stoichiometry. Predicted stoichiometries are shown on the right- or left-hand y axes for the DC with wild-type or M3 AXIN1, respectively. Dashed lines indicate mean number average molecular weight (M_n) values from SEC-MALS; shaded areas indicate corresponding 95% confidence intervals.