For Figure 1, our procedure for obtaining cytoplasmic and nuclear protein fractions has been described previously (29 (link)). To obtain the insoluble protein fraction, the pellet that was not soluble in either the hypertonic or nuclear lysis buffers was resuspended in the same volume of cell lysis buffer as used to obtain soluble protein fractions (550 μl). The 10% Bis–Tris NuPage Gel (Invitrogen) was loaded with 20 μg of soluble protein and 40 μl of insoluble suspension, transferred via iBLOT (Invitrogen) to a nitrocellulose membrane, immunoblotted for HA and β-actin, and imaged as previously described (29 (link)) except that rat α-HA (3F10, Roche) diluted 1:1000 and goat anti-rat 800 diluted 1:15 000 were used to detect HA. For Figure 7, cells were harvested by scraping in PBS, washed once in PBS, and frozen. RIPA (Sigma) supplemented with PhosSTOP (Roche) and cOmplete ULTRA Tablets (Roche) inhibitors was added to each thawed cell pellet and cells were further lysed by freeze-thaw. Protein lysates were analyzed by Pierce BCA Protein Assay Kit (Thermo Scientific) and 25 μg protein was combined with NuPAGE sample buffer and reducing agent (Life Technologies), loaded onto 4–12% Bis–Tris gels (Life Technologies), and processed further as described above. Image Studio software (LiCOR) was used for quantification and further analysis was performed in Excel and GraphPad Prism.
For detection of Flag, the buffers and washes were Tris and milk-based as described elsewhere (30 (link)). The amount of unpurified protein sample loaded was 30 μl while the amount of purified protein sample loaded was 20 μl. The primary rabbit α-Flag antibody was diluted 1:1000 (Cell Signalling, Danvers, MA) and the secondary antibody was goat anti-rabbit HRP diluted 1:10 000 (Bio-Rad, Hercules, CA). The membrane was developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Pierce) and imaged on a ChemiDoc XRS+ (Bio-Rad). The membrane was stained with Ponceau S Staining Solution (Tocris Biosciences, Bristol, UK).