Tnt coupled in vitro transcription translation system

GST-PGC-1α vector was a gift from Pere Puigserver, and was transformed into BL21 (NEB) to express the protein. IRF4 was synthesized using a TNT-coupled in vitro transcription/translation system (Promega). The resultant protein was incubated with GST-PGC-1α and then immobilized on glutathione-Sepharose beads according to the manufacturer’s instructions (Thermo). Proteins were eluted by glutathione and analyzed by western blotting.

In vitro translated proteins were synthesized and 35S-labeled using the TNT coupled in vitro transcription/translation system (Promega) from Sp-Elk WT linearized construct under the control of a T3 promoter. 35S-labeled proteins were separated by NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and identified by autoradiography. Recombinant Sp-Elk His-tagged protein was expressed in E. coli and purified Ni⁺² affinity chromatography. 1µg recombinant protein was phosphorylated in a reaction that contained kinase buffer (4 mM MOPS; 1 mM EGTA; 0.2 mM DTT; 0.2 mM NaF; 15 mM MgCl2; 2mM DTT, 5 mM β-glycerol phosphate), 100 uM ATP, and 2ng/µl activated recombinant ERK1 kinase (Upstate Biotechnology, Lake Plaid, NY) in a 50ul reaction.