Author: Renata C Fleith; Harriet V Mears; Edward Emmott; Stephen C Graham; Daniel S Mansur; Trevor R Sweeney
Title: IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA Document date: 2018_2_8
ID: j97gul0w_22
Snippet: Stable isotope labelling with amino acids in cell culture and immunoprecipitation HEK293T cells were cultured in Arg/Lys-free DMEM, supplemented with light (R0K0), medium (R6K4) or heavy (R10K8) amino acids, as described (32) . 1x10 7 cells were transfected with 10 μg plasmid DNA using Lipofectamine 2000 (ThermoFisher). After 24 hours, media was replaced to contain 1000 U/mL human interferon ï¡-2a (Roferon-A, Roche) for a further 16 hours. Cell.....
Document: Stable isotope labelling with amino acids in cell culture and immunoprecipitation HEK293T cells were cultured in Arg/Lys-free DMEM, supplemented with light (R0K0), medium (R6K4) or heavy (R10K8) amino acids, as described (32) . 1x10 7 cells were transfected with 10 μg plasmid DNA using Lipofectamine 2000 (ThermoFisher). After 24 hours, media was replaced to contain 1000 U/mL human interferon ï¡-2a (Roferon-A, Roche) for a further 16 hours. Cells were harvested in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 % Triton X100) containing 1:200 Protease Inhibitor Cocktail Set III (Merck) and 1:200 Benzonase nuclease (Sigma-Aldrich). Lysates were normalised to 3 mg/mL of protein before incubation with anti-FLAG-M2 affinity gel (Sigma) at 4 ï‚°C for 18 hours. Beads were washed 3 times in Tris-buffered saline, then resuspended in 2X SDS-sample buffer (Invitrogen) and boiled for 5 minutes to elute bound proteins.
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