Realtime 2-5A kinetics suggests interferons ß and ? evade global arrest of translation by RNase L

Alisha Chitrakar; Sneha Rath; Jesse Donovan; Kaitlin Demarest; Yize Li; Raghavendra Rao Sridhar; Susan R. Weiss; Sergei V. Kotenko; Ned S. Wingreen; Alexei Korennykh

Normally, RNase L is activated as a part of the innate immune system. However, RNase L activation by small molecules could be explored for developing adjuvants and anticancer therapeutics with some of the beneficial effects of mTOR blockers, and with an added advantage of maintaining the protein synthesis activity of the innate immune system. The search for such on demand activators can be facilitated by biochemical, cell-based as well as in vivo applications of the 2-5A biosensor we describe here. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/476341 doi: bioRxiv preprint (v/v) glycerol, 5 mM DTT and 1% Triton X-100. Crude lysates were centrifuged at 35,000 g for 30 minutes. Clarified lysates were affinity purified using glutathione sepharose (GE Healthcare Life Sciences) and the GST tag was removed with Prescission Protease (GE Healthcare Life Sciences). Protein concentration was determined by band densitometry of coomassie-stained SDS PAGE gels using bovine serum albumin as a standard.