Cell lysate preparation

The cells were removed from the plates by scraping after washing once with PBS (phosphate buffered saline), after which cells were collected by centrifugation (14K rpm, 14 seconds), washed again with ice cold PBS followed by another round of centrifugation and resuspended in a volume of TNESV lysis buffer (50 mM Tris-HCl, pH 7.4; 1 % NP-40; 2 mM EDTA, pH 8.0; 0.1 M NaCl) containing protease (Roche, Penzberg, Germany) and phosphatase inhibitors (Sigma-Aldrich). In instances that cell lysates were used for cap-affinity assays, cells were resuspended in a volume of freeze-thaw lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 10 mM tetrasodium pyrophosphate) containing the same protease and phosphatase inhibitors followed by three freeze-thaw cycles (15 min at −80°C, 2 minutes at 37°C). Protein lysate concentration was determined by Bradford assay (Bio-Rad, Hercules, CA) and stored at −80°C.