Zirconium silicate beads

To prepare cell lysate, cells were washed with ddH2O supplemented with 0.2 mM PMSF and resuspended in 1 mL/g sample of buffer H (25 mM HEPES pH 8.0, 2 mM MgCl2, 0.1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.1% IGPAL, 150 mM KCl) with protease inhibitors cocktail (Price) plus 3.6 g/g sample of Zirconium Silicate beads (Next Advance). Cells were homogenized in the Blue Bullet Blender at speed 10 for 15 min in a cold room, and the lysate was cleared by centrifugation at 70000 g for 30 min at 4°C. For immunoprecipitation, 15 μg/g sample of anti-HA or 12 μg/g sample of anti-Myc antibodies were added to the lysates, incubated overnight at 4°C in rotation followed by incubation with 30 μL/g sample of Dynabeads M-280 Sheep Anti-Rabbit or Anti-Mouse IgG for 1 h at 4°C with rotation. Beads were washed four times with ice-cold IP buffer, and bound proteins were eluted by boiling for 5 min with SDS-PAGE sample buffer. Proteins were separated by 4%–15% gradient SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. The blots were probed with individual primary antibodies as indicated, and then incubated with HRP-conjugated donkey anti-rabbit or anti-mouse antibodies. In all blots, proteins were visualized by enhanced chemiluminescence.