IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA

Renata C Fleith; Harriet V Mears; Edward Emmott; Stephen C Graham; Daniel S Mansur; Trevor R Sweeney

The raw data files were processed and quantified using MaxQuant v1.5.7.4 (33) and searched against the Uniprot Human database (70,550 entries, dated 19th September 2016) using the built-in Andromeda search engine. Peptide precursor mass tolerance was set a 4.5 ppm, and MS/MS tolerance was set at 0.5 Da. Search criteria included carbaminomethylation of Cys as a fixed modification. Oxidation of Met and N-terminal acetylation were selected as variable modifications. Quantification was based on Light (Arg 0, Lys 0), Medium (Arg 6, Lys 4), and Heavy (Arg 10, Lys 8) SILAC labels. Searches were performed with tryptic digestion, a minimum peptide length of seven amino acids, and a maximum of two missed cleavages were allowed. The reverse database search option was enabled and the maximum false discovery rate for both peptide and protein identifications was set to 0.01. Quantitation was performed using a mass precision of 2 ppm. The full MaxQuant output is provided as part of PRIDE submission PXD007584 permitting viewing of annotated spectra in MaxQuant v1.5.7.4. Downstream analysis was accomplished in the Perseus software (34) . Contaminants and reverse database hits were removed, and protein ratios were log2transformed. Proteins were considered to represent putative interaction partners if they showed a significant (t-test, p<0.05) increase in their abundance compared with the control pulldown and had to have been identified in at least two of the three replicates.