*2.9. GST Pulldown Assay*

HEK293FT cells were transfected using Polyethylemine (PEI; Polysciences Inc.) as previously described [33]. GST pulldown assays were performed by incubating for 16h at 4 ◦C Glutathione Sepharose 4B (GE Healthcare) with whole cell lysates prepared 48 h after transfection. The resin was extensively washed with 'mild' Radioimmunoprecipitation Assay (RIPA) buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1 mM Ethylenediaminetetraacetic acid, 1% NP40, and 2.5 mM Dithiothreitol), boiled in loading buffer, and analyzed by Western blotting using anti-GST (Santa Cruz Biotechnology; B-4) and anti-GFP (Millipore; MAB3580) antibodies.
