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. 2013 Oct 3;9(10):e1003663. doi: 10.1371/journal.ppat.1003663

Figure 2. Human and mouse IFIT1 bind directly to unmethylated capped RNA.

Figure 2

(a) Isolation of luciferase-tagged human IFIT (hIFIT) proteins from transfected 293T cells with beads coated with 250 ng RNA bearing 5′ OH, PPP or CAP. The graphs show luciferase activity after affinity purification (AP) with PPP-RNA and CAP-RNA (normalized to OH-RNA) and the activity of 10% of the input lysates. (b) Data obtained (as in a) for luciferase-tagged murine Ifit (mIfit) proteins affinity purified with PPP-RNA and CAP-RNA. (c) Recombinant His-tagged hIFIT1, -2, -3, and -5 were incubated with beads only or beads coated with OH-RNA or CAP-RNA. Bound proteins were detected by western blotting. Input shows 1/10th of the amount incubated with beads. (d) Purification of luciferase-tagged wild-type (WT) and hIFIT1 mutants with CAP-RNA-coated beads. The graphs show luciferase activity after affinity purification and the activity of 10% of the input lysates. (e) Ratios of LFQ intensities of proteins identified by mass spectrometry in precipitates of CAP-RNA vs. OH-RNA in IFN-α-treated MEFs from wild-type (Ifit1+/+, grey bars) and Ifit1-deficient (Ifit1−/−, black bars) C57BL/6 mice. Error bars indicate means (±SD) from three independent affinity purifications. Asterisks indicate ratios with negative values.