Glutathione (gsh)

Cell pellets or tissues were homogenated with TNEN buffer (50 mM Tris-HCl 8.0, 150 mM NaCl, 5 mM EDTA and 1% NP-40) with protease inhibitors (Roche, USA) and phosphatase inhibitors (Roche, USA) and centrifuged at 14,000 × g for 10 min at 4 °C. For co-immunoprecipitation, the supernatant was mixed indicated antibodies overnight at 4 °C and then with 20 μl Protein G Dynabeads (Life technologies, Carlsbad, CA) for 2 h at 4 °C. For GST pulldown, GST, or GST-tagged proteins were precipitated for 2 h at 4 °C with Glutathione Sepharose (GE, Milwaukee, WI), and eluted with 10 mM Glutathione reduced solution following three times washing with TNEN buffer. Proteins were separated by SDS-PAGE gel and transferred to PVDF membranes. After blocking, membranes were probed with various primary antibodies and then HRP-linked secondary antibodies. Signals were detected using the ECL method.

Full-length 6His-FIP5 and 6His-CGN were produced in baculovirus using the transfer vector pVL1392 (ref. 8 (link)). In brief, 106 Sf9 cells were seeded into a six-well plate, and the Bacfectin–DNA mixture was added dropwise. After 5 days, the P1 viral stock was harvested and further amplified to P2 and P3 stages. For protein production, 1 l of Sf9 cells at 2 million cells per ml was infected with 2 ml of P3 viral stock (approximate MOI of 0.5) and harvested after 65 h. Cells were lysed in 50 mM Tris buffer, pH 7.5, containing 300 mM NaCl, and the cleared lysate was loaded onto a Ni-NTA column. Eluted 6His-FIP5 or 6His-CGN was dialysed overnight against buffer (50 mM Tris, pH 7.5, 300 mM NaCl and 5 mM BME) and frozen in liquid nitrogen. Yields were typically 3–5 mg l⁻¹ with an estimated purity of >75%. GST-CGNaa1-406, GST-CGNaa355-579, GST-CGNaa571-794 and GST-CGNaa781-1025 fragments were expressed using the pGEX-4T plasmid (provided by Sandra Citi18 (link)) and purified using the BL21-(FE3) RIPL Escherichia coli strain8 (link). Briefly, E. coli were lysed using a French press and then incubated with glutathione agarose beads (Sigma-Aldrich). Beads were then washed with PBS and the GST-protein was eluted with 25 mM glutathione (GE Healthcare). Final protein concentrations were determined using Bradford protein assay (Bio-Rad Laboratories).