In vitro interaction of TACC2 and RXRβ3
The TACC2 cDNA was cloned into GST fusion vector pGEX5X2 (Amersham Biosciences, Piscataway, NJ, USA). GST and GST-TACC2 proteins were expressed in E. coli BL21(DE3) plys "S" with 1 mM IPTG at 37°C shaker for 2 hrs. Cells (50 ml) were harvested and resuspended in 5 ml of 20 mM Tris-HCl pH.8.0, 200 mM NaCl, 1 mM EDTA pH8.0, Protease inhibitor set III (Calbiochem). The cells were lysed by sonication and lysate cleared by centrifugation at 7500 rpm at 4°C for 15 min. The cleared lysate was immobilized on glutathione sepharose beads in 3 ml of 20 mM Tris-HCl pH.8.0, 200 mM NaCl, 1 mM EDTA pH8.0). RXRβ3 cDNA was cloned into pET 28C(+) (Invitrogen, Carlsbad, CA, USA) and protein synthesized by TNT quick coupled transcription/translation system kit (Promega) and radiolabeled with 35S methionine according to manufacturer's instructions. 100 μl of in vitro translated RXRβ3 protein in 1 ml of 20 mM Tris-HCl pH.8.0, 200 mM NaCl, 1 mM EDTA pH8.0 was incubated at 4°C with immobilized GST-TACC2 or GST for 90 min. Unbound RXRβ3 was removed by washing three times with 20 mM Tris-HCl pH.8.0, 200 mM NaCl, 1 mM EDTA pH8.0. Bound proteins were eluted from the beads at room temperature for 10 min in elution buffer (100 mM Tris HCl, pH8.0, 20 mM reduced glutathione). The proteins were analyzed on 12% SDS polyacrylamide gels. Coomassie blue staining verified equal loading of GST fusion protein. Dried gels were autoradiographed.