Single-stranded VSV leader RNA (nucleotides 1-18) was T7 polymerase-transcribed in presence of [a-32 P]-CTP, yielding radiolabeled 59-triphosphorylated (ppp-) RNA, followed by alkaline phosphatase treatment for generation of 59-hydroxyl (HO-) RNA. ppp-RNA or HO-RNA were added to bacterially expressed and purified 6xHis-tagged Ifit1 or Ifit2 protein in reaction buffer (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.05% Triton X-100, 10% glycerol) and incubated for 30 min on ice. Reaction products were separated by 6% native polyacrylamide gel electrophoresis followed by exposure to film.