PMC:441373 / 48297-49764 JSONTXT

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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. 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