Concerted 2-5A-Mediated mRNA Decay and Transcription Reprogram Protein Synthesis in dsRNA Response

Sneha Rath; Eliza Prangley; Jesse Donovan; Kaitlin Demarest; Yigal Meir; Ned Wingreen; Alexei Korennykh

Frozen A549 cell pellets (~200 L) were resuspended in 500 L of 20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 5 mM MgCl 2 , 4 mM DTT, 0.2% NP-40, 1x phosphatase inhibitors 2/3 (Sigma), 2x complete protease inhibitor (Roche), and 0.4 U/ml RNase inhibitor (NEB), as described previously (Lorsch and Herschlag, 1999) . Resuspended cells were rotated for 15 minutes at 4°C, followed by centrifugation for 20 minutes at 16,000 x g, 4°C. Obtained supernatants were centrifuged for 30 min at 21,000 x g, 4°C. The resulting supernatants were centrifuged for 80 minutes at 300,000 x g and 4 °C to yield a crude ribosomal pellet. Pellets were washed with 5 mM HEPES-KOH (pH 7.5), 50 mM KCl, 1.5 mM MgCl 2 , 4 mM DTT and resuspended in 100 L of the fresh wash buffer.