Purified Dvl2 DIX filament samples with or without Axin DAX were incubated at room temperature for 30 min, then centrifuged at 386,000 x g for 7 min at 4°C. After removal of the supernatant, the pellet was resuspended by vortexing in 1X SDS-PAGE sample buffer for 30 s. Pellet and supernatant fractions were then run on a 15% Tris-glycine-SDS gel containing 6M urea. The gel was stained with Coomassie blue and imaged on a LiCOR scanner (LICOR, Inc, Lincoln, NE), and band intensities determined in FIJI (Schindelin et al., 2012 (link)). Data from biological and technical replicates were analyzed in GraphPad PRISM 8. Error bars represent the standard error of the mean.
For filament formation assays, MBP-DIX at the indicated concentrations was incubated with 27 ng/µL TEV protease overnight at 4°C, and the samples were then pelleted as described above. Supernatant and pellet samples were run on a NuPage 4–12% Bis-Tris gel (ThermoFisher Scientific), which was visualized with Sypro Ruby stain (ThermoFisher Scientific, Waltham, MA) using a BioRad Gel Doc EZ Gel Imager (BioRad, Hercules, CA). Band intensities were quantified and plotted as described above.
For filament formation assays, MBP-DIX at the indicated concentrations was incubated with 27 ng/µL TEV protease overnight at 4°C, and the samples were then pelleted as described above. Supernatant and pellet samples were run on a NuPage 4–12% Bis-Tris gel (ThermoFisher Scientific), which was visualized with Sypro Ruby stain (ThermoFisher Scientific, Waltham, MA) using a BioRad Gel Doc EZ Gel Imager (BioRad, Hercules, CA). Band intensities were quantified and plotted as described above.