Gateway compatible modified ptnt vectors

For in vitro pull-down assays, additional constructs were made. The pENTR/D-TOPO plasmids (Invitrogen) containing the sequences encoding GI, PIF1, and PIF5 have been previously described (Pruneda-Paz et al., 2014 (link); Sawa and Kay, 2011 (link)). Full-length PIF3, PIF4, and GFP sequences were amplified by PCR (primers used are listed in supplemental Table S2) and cloned into the pENTR/D-TOPO vector (Invitrogen). Partial PIF3 sequences were amplified by PCR (primers used are listed in supplemental Table S2) and cloned into the pDONRZeo vector (Invitrogen) by Gateway BP recombination reaction (Invitrogen). 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) (Nito et al., 2013 (link)), which were kindly provided by Dr. Joanne Chory (The SALK Institute, La Jolla, CA). The vectors contained an N-terminal HA or Flag tag as specified in each case. 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 (Pedmale et al., 2016 (link)) using anti-HA 3F10 antibody (Roche).