Assays

Frozen cell pellets from 4x15 cm cell culture plates were lysed in Glycerol buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.5 % Triton-X-100, 10 % Glycerol, 1x protease inhibitor, 1x phosphatase inhibitor) for 30 min at 4° C with end-over-end rotation. Lysates were cleared from cell debris by centrifugation prior to adjustment of protein concentrations between the samples and overnight immunoprecipitation at 4° C with pre-equilibrated anti-HA-agarose (Sigma). Agarose beads were washed five ...