For in vitro assays, purified recombinant Nab2-N (amino acids 1–97) and CT-Mlp1 (amino acids 1490–1779) were employed. Nab2-N was expressed as a tobacco etch virus protease-cleavable GST fusion protein to assess direct binding.37 GST-Nab2-N (pAC2058) was expressed in E. coli DE3 cells. Cells were collected and lysed in phosphate-buffered saline (PBS) (137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, pH 7.4) supplemented with protease inhibitor mixture (1 mM PMSF, 3 ng/ml pepstatin A, leupeptin, aprotinin, and chymostatin) by incubation with lysozyme (0.1 mg/ml) for 30 min on ice followed by sonication to purify the GST fusion proteins. Lysates were clarified by centrifugation and incubated with glutathione Sepharose (Amersham) in buffer A [20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM β-mercaptoethanol, 1 mM PMSF, and 0.1% Igepal] for 2 h at 4 °C with mixing. The beads were then washed with PBS and 0.5% Triton X-100.