Tnt t7 quick coupled in vitro transcription translation system

³⁵S-labeled FoxO1 protein was synthesized by using the TNT-T7 quick-coupled in vitro transcription/translation system (Promega). GST tagged human HMGA1 was obtained using the pcDNA1-GST/HMGA1 expression vector, a kind gift from D. Thanos (Institute of Molecular Biology, Genetics and Biotechnology, Athens, Greece)^{24 (link)}. Covalent coupling of antibody to protein A-Sepharose (GE Healthcare) was performed as previously described^{67 (link)}. Antibody-coupled protein A beads were washed twice in phosphate-buffered saline and used in immunoprecipitation studies. Briefly, aliquots of HepG2 cell nuclear extract or pure HMGA1 were incubated for 3 h with rotation at 4 °C with 10 µl of antibody coupled protein A beads. Beads were recovered by gentle centrifugation and washed three times with 500 µl of NETN wash buffer [50 mM Tris-HCl (pH 8.0), 0.1% NP-40, 150 mM NaCl, 1 mM EDTA] for 5 min. Protein was removed from the beads by boiling in sample buffer for 5 min and analyzed by SDS-PAGE and immunoblotting^{67 (link)}. Antibodies used for these studies were as follows: anti-HMGA1^{24 (link)} and anti-FoxO1 (sc-11350) (Santa Cruz Biotechnology).

The ARF6-Myc, HA-RGA and HA-YFP proteins were synthesized by TNT T7 Quick Coupled in vitro transcription/translation system (Promega). The ARF6-Myc proteins were pre-incubated with anti-Myc antibody (Cell Signaling Technology)-bound protein A-Dynabeads (Life Technology) for 2 hr. After removing unbound ARF6-Myc proteins, the HA-RGA or HA-YFP proteins were incubated with the ARF6-Myc-bound Dynabeads for 1 hr in PBSN buffer (PBS buffer + 0.1% NP-40). The beads were washed three times with the PBSN buffer and the pulled-down proteins were analyzed by immunoblots using anti-HA antibody (Roche) and anti-Myc antibody (Cell Signaling Technology).