To express proteins in the cell-free system, all inserts were transferred by Gateway LR recombination reaction (Invitrogen) into Gateway compatible modified pTnT vectors (Promega) containing an N-terminal HA or FLAG tag as specified in each case. pDONR207-PIF7 was recombined with the pTnT-HA vector to produce PIF7 fused to HA. The construct containing GI in the pTnT-FLAG vector has already been described (19 (link)). For the in vitro pull-down assays, proteins were co-expressed using TnT® SP6 High-Yield Wheat Germ Protein Expression System (Promega) as per manufacturer’s instructions. Five percent of the reactions (2.5 µl) were used to verify expression of the proteins (input) and the remaining extract was immunoprecipitated as earlier described (47 (link)) using a modified IP buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1x protease inhibitor cocktail (Roche), 1x Phosphatase Inhibitors I&II (Sigma) and 25 μM MG-132) and the anti-HA 3F10 antibody (Roche).