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

Differential scanning fluorimetry experiments to determine the melting temperature (T M ) of IFIT2, IFIT3 and IFIT2:IFIT3 were performed using a Viia7 Real-Time PCR system (Applied Biosystems). In an optical 96-Well Reaction Plate (Applied Biosystems 4366932), 1:500 Protein Thermal Shift dye (Life Technologies, 4461146) was mixed with 0.1 mg/mL protein in a final buffer composition of 20 mM HEPES pH 7.5, 150 mM KOAc, 2.5 mM MgOAc, 5% glycerol, and 1 mM DTT in a final volume of 20 μL. Emission from quadruplicate samples was measured at 623 nm while ramping from 25 -95 C stepwise at a rate of 1 C per 20 seconds. To determine Tm, data were analysed by non-linear regression using the Boltzmann equation y = LL + (UL -LL)/(1 + exp(Tm -x)/a) where LL and UL are the minimum and maximum fluorescence intensities, respectively (31) .