Pull-down assays using Arabidopsis extracts and GST-fusion proteins produced in E. coli were carried out as described previously20 (link). Recombinant GST, GST-BZR1, GST-PIF3 and GST-PIF4 bound to glutathione sepharose beads were used separately to pull-down FLAG-RGA from protein extracts from PRGA:FLAG-RGA transgenic Arabidopsis in SPY sly1-10 rga-24 or spy-8 sly1-10 rga-24 background.
For RT-qPCR analysis, total RNA was isolated from 9-day old seedlings of ga1-3, ga1-3 spy-8 and ga1-3 spy-19 using a Quick-RNA MiniPrep kit (Zymo Reseaarch). First strand cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science). For qPCR, the FastStart Essential DNA Green Master mix was used on a LigthCycler 96 Instrument (Roche Applied Science).
For RT-qPCR analysis, total RNA was isolated from 9-day old seedlings of ga1-3, ga1-3 spy-8 and ga1-3 spy-19 using a Quick-RNA MiniPrep kit (Zymo Reseaarch). First strand cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science). For qPCR, the FastStart Essential DNA Green Master mix was used on a LigthCycler 96 Instrument (Roche Applied Science).
