Tnt t7 quick coupled transcription translation kit

Full length SOX9‐WT and SOX9‐T236&240A was synthesized in vitro using TnT T7 Quick Coupled Transcription/Translation kit from Promega (L1170) according to the manufacturer's instructions. Following the synthesis, the proteins were immunoprecipitated and immobilized on Sepharose beads prior to being used for either in vitro kinase or binding assay. For the kinase assay, the immobilized SOX9 proteins were incubated with a complete GSK3 reaction buffer for 90 min at 37°C. Complete GSK3 reaction buffer consists of 50 mM Tris–HCl pH 7.4, 1 mM EGTA, 150 mM NaCl, 0.1% β‐mercaptoethanol, 2 mM ATP, and 1 U of recombinant, active human GSK3β (Abcam, AB60863). For in vitro binding assay, Sepharose bead‐immobilized full‐length SOX9 protein or synthetic peptide was resuspended in 50 mM Tris–HCl pH 7.4 and 150 mM NaCl buffer in the presence of recombinant, active SCF^FBW7 complex (Millipore, 23‐030) under constant gentle agitation at 37°C for 1 h. Following the incubation, the supernatant was aspirated and the beads were washed three times with NP‐40 lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP‐40) prior to elution under reducing–denaturing condition in SDS sample buffer at 95°C for 5 min for SDS–PAGE.