Detergent-Based Protein Fractionation Assay

For the solubility assays, cell lysis and protein fractionation based on detergent solubility were performed as previously described (Figure 2A; Guo and Lee, 2011 (link); Kfoury et al., 2012 (link); Silva et al., 2016 (link)). Briefly, higher solubility proteins (S fractions) were purified in 1% Triton buffer [1% Triton X-100 (Thermo Fisher Scientific), 1% Halt Protease/Phosphatase inhibitors (Thermo Fisher Scientific), 1:5,000 Benzonase (Sigma) and 10 mM DTT (New England BioLabs) in DPBS], whereas lower solubility pelleted proteins (P fractions) were resuspended in 5% SDS buffer [5% SDS (Sigma), 1% Halt Protease/Phosphatase inhibitors (Thermo Fisher Scientific), 1:5,000 Benzonase (Sigma) and 10 mM DTT (New England BioLabs) in RIPA buffer]. SDS-PAGE western blot was performed by loading 20 μg of each S-fraction and equal volume of the P-fraction onto pre-cast Tris-Acetate SDS-PAGE (Novex, Invitrogen). Western blot was performed as before. Densitometry values (pixel mean intensity in arbitrary units, a.u.) were measured with the Histogram function of Adobe Photoshop 2021 (v.22.4.3) and normalized to the respective GAPDH intensity in the S-fraction. Calculations were done in Microsoft Excel (v.16.52) and graphs were plotted in GraphPad Prism 9 (v.9.2.0).