Purification and Characterization of ARX Transcription Factor

Sequences corresponding to amino acids 220–392 of ARX^WT/mut were amplified from pEGFP-C1-Arx^WT/mut as described above and then cloned into the vector of pGEX-4T-2 (GE Healthcare Life Sciences). GST-ARX (220–392)^WT/mut protein was expressed in Escherichia coli BL21 competent cells (Invitrogen), and its expression was induced by the addition of 0.2 mM IPTG to late logarithmic cultures (OD = 0.6) for 4 h at 25°C. Cells were then harvested, resuspended in bacteria lysis buffer (50 mM Tris/HCl [pH 8.0], 200 mM NaCl, 200 mM EDTA, 10% Glycerol, 1 mM DTT, 1 mM PMSF, 1 mg/ml lysozyme) and disrupted by sonication. After centrifugation, the supernatants were applied to Glutathione Sepharose 4B beads (GE Healthcare Life Sciences), and the beads were washed with PBS. The GST fusion protein was finally eluted with 30 mM reduced glutathione in 50 mM Tris-Cl, pH 8.0.
Electrophoretic mobility shift assays (EMSAs) were performed as previously described with slight modifications [24 (link)]. The purified ARX protein truncation was applied to 6% polyacrylamide gel with double-stranded oligonucleotides, SIX1, F (5′-TCTTAACATTAAGGTAATTAAATATGAGCTCAC-3′)/SIX1, R (5′-GTGAGCTCATATTTAATTACCTTAATGTTAAGA-3′) labeled by biotin (Sangon Biotech). To detect the DNA/ARX complex, we utilized the LightShift® Chemiluminescent EMSA kit (Thermo Scientific).