Probing PAK4-PPARγ Protein Interactions

The PAK4 truncated mutants were synthesized in vitro with specific PCR-amplified fragments using Transcend Biotin-Lysyl-tRNA and TnT Quick Coupled Transcription/Translation System (Promega, Madison, WI) following the manufacturer’s instructions. The recombinant GST and GST-tagged PPARγ fusion protein was expressed bacterially following induction with IPTG (1.0 mg/ml) and was purified using MagneGST Pull-Down System (Promega) following manufacturer’s protocol and verified by SDS-PAGE. About ~2 µg aliquots of protein-coated GST particles were incubated with biotin-labeled PAK4 truncated mutants overnight at 4°C. Beads were washed for 2–3 times, eluted in 20 µl of pre-heated sample buffer, separated on SDS–PAGE, transferred to nitrocellulose membrane. The in vitro protein binding was detected with Transcend Nonradioactive Translation Detection Systems following manufacturer’s instructions.^{26 (link)}