Cells in 140 mm dishes were washed with PBS twice before the addition of PBS containing 1 mM CaCl2 and 1 mM magnesium acetate. The crosslinking reagent DSP [dithiobis(succinimidyl propionate)] (ThermoFisher) dissolved in DMSO was then added to the cells to a final concentration of 0.5 mM and crosslinking allowed to proceed for 20 min at room temperature. The cells were then washed twice with PBS containing 5 mM Tris-HCl before lysis. For immunoprecipitation experiments, cells were harvested from 140 mm tissue culture dishes using a cut rubber bung to scrape the cells into lysis buffer (20 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 200 mM sorbitol and 2 mM EDTA with 0.1% Triton X-100 for the native immunoprecipitation and Tris-buffered saline with 0.5% Triton X-100 for the non-native immunoprecipitation following crosslinking). The lysate was centrifuged at 10,000 g for 5 min to pellet insoluble material and then incubated with Protein-A Sepharose for 30 min as a preclearing step. Following a second spin at 10,000 g for 5 min, the lysate was then treated with antibodies against the target
