Sypro ruby stain

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 analysis of the phospho-proteome, cells were plated and irradiated with 10 Gy SD, or with 10 fractions of 1 Gy dose per fraction with two fractions per day (Fig. 1A). At 30 min (ST) and at 2 months (LT) after irradiation, the cells were lysed from plates in T-Per (ThermoFisher Scientific) mixed 1:1 with 2X SDS Tris–Glycine buffer (Invitrogen, Carlsbad, CA) + 2-mercaptoethanol (final concentration = 2.5%). Reverse phase protein microarrays were performed as previously published^{22 (link)}. In brief, samples were diluted and printed in duplicates onto nitrocellulose slides. HeLa cell lysates (with or without pervanadate) were used as positive and negative controls (Supplementary Figure S2). Microarrays were stained with specific and validated antibodies and analyzed with a biotin-linked signal amplification system (DAKO). The total protein amount of the sample was determined with the SYPRO Ruby stain (ThermoFisher Scientific).

To assess the purity of γ-TuRC, 3–5 μl of purified γ-TuRC was visualized on an SDS-PAGE with SYPRO Ruby stain (ThermoFisher) following the manufacturer’s protocol. Biotinylated subunits of γ-TuRC were assessed by immunoblotting with Streptavidin-conjugated alkaline phosphatase (S921, ThermoFisher). For further conjugation of Alexa-568 dye to γ-TuRC, fluorescently labeled subunits were assessed by visualizing an SDS-PAGE gel with Typhoon FLA 9500 (GE Healthcare) with LPG filter and 100 μm pixel size. γ-TuRC purification was also assessed by visualizing using electron microscopy. 4 μl of peak sucrose gradient fraction of γ-TuRC was pipetted onto CF400-Cu grids (Electron Microscopy Sciences), incubated at room temperature for 60 s and then wicked away. 2% uranyl acetate was applied to the grids for 30 s, wicked away, and the grids were air-dried for 10 min. The grids were imaged using Phillips CM100 TEM microscope at 64,000x magnification.

ID93 concentration was quantified after reconstitution back to the feedstock concentration using densitometry analysis of reducing SDS-PAGE based on a standard curve. Samples were prepared by mixing a 20% (w/v) sodium dodecyl sulfate solution (Thermo Fisher Scientific, Waltham MA, United States), 4X LDS Buffer (Thermo Fisher Scientific, Waltham, MA, United States) spiked with 5% (v/v) β-mercaptoethanol, and reconstituted sample in a 2:1:1 ratio. Due to the elevated ID93 concentration, prepared Group 5 (V-D-N) samples were diluted 1:50 to bring ID93 concentration into the range of the standard curve. The upper limit of quantitation of the assay is 0.02 mg/ml ID93. Samples were heated for 15 min at 85°C and loaded into a 4–20% Tris-Glycine SDS-PAGE gel (Thermo Fisher Scientific, Waltham, MA, United States). The gel was run at 180V for 65 min and then stained overnight using a SYPRO Ruby stain (Thermo Fisher Scientific, Waltham, MA, United States) and imaged (ChemiDoc; Bio-Rad, Mississauga, ON, Canada). ID93+GLA-SE standards at 10 ng, 50 ng, and 100 ng protein load were prepared in the same manner and included on each gel. Densitometry analysis was performed using Image Lab 6.0 software (Bio-Rad Laboratories, Hercules, CA, United States). The three standards were used to generate a standard curve based on band intensity and ID93 was interpolated from the standard curve.