To perform UV-induced crosslinking assay, hPGI (0.7 μg) was mixed with 1 μCi [α-32P]GTP (3000 Ci/mmol/) in an 18 μl solution and placed on ice for 20 min. The reaction mixture was irradiated with UV light (254 nm) for 2 min at a distance of 8 cm using CL1000 ultraviolet crosslinker (UVP, Upland, California). The irradiated product was separated on a 12% SDS-polyacrylamide gel, followed by autoradiography using BAS-2500 Phosphorimager (Fujifilm, Tokyo, Japan).
For GTP-Sepharose binding assay, hPGI (~1 mg) was load onto a 1 ml GTP-Sepharose column (Jena Bioscience, Jena, Germany) that had been equilibrated with 40 mM TRICINE buffer, pH 7.5. After extensive wash with the equilibrium buffer, the protein bound to the column was eluted with the equilibrium buffer that contained 5 mM GTP. The presence of hPGI in every fraction was analysed by SDS-12% PAGE. A parallel experiment using a Sepharose column was conducted as the control.
For GTP-Sepharose binding assay, hPGI (~1 mg) was load onto a 1 ml GTP-Sepharose column (Jena Bioscience, Jena, Germany) that had been equilibrated with 40 mM TRICINE buffer, pH 7.5. After extensive wash with the equilibrium buffer, the protein bound to the column was eluted with the equilibrium buffer that contained 5 mM GTP. The presence of hPGI in every fraction was analysed by SDS-12% PAGE. A parallel experiment using a Sepharose column was conducted as the control.