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

To generate puromycin-tagged nascent peptides, human cells were treated with 0.1-1 µg/mL of poly-IC for times specified on the figures, after which the growth media was supplemented with 10 µg/mL puromycin (Invitrogen). Puromycin pulse lasted for 5 min. Cells were trypsinized and harvested in NuPAGE LDS sample buffer for western blot analyses. Proteins were separated by 10% BisTris PAGE (NuPAGE), and transferred on PVDF membranes (Life Technologies). The membrane was stained with Ponceau to normalize for sample loading, then washed and blocked with 5% non-fat dry milk in TBST buffer. The membranes were probed with 1:1000 mouse anti-puromycin antibody (EMD Millipore) that binds to de novo synthesized proteins, followed by horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:10,000, Jackson ImmunoResearch).