Characterization of TLX-DNA Interactions

The synthetic oligonucleotides (5′-GCC CAT AGC CAT CAG TCA CAA CTA CCA-3′) containing one TLX-binding site (5′-CAG TCA-3′) were annealed and labeled with ³²P-dCTP as a probe. Gel-shift assays were performed essentially as previously described (Zhang et al., 2006 (link); Qin et al., 2013 (link)). Briefly, in vitro translated TLX protein was incubated with ³²P-labeled probes (5 × 10⁵ c.p.m.) in binding buffer for 20 min at RT. Antibody-dependent supershift assays were performed by adding 1 μg rabbit anti-HA antibody (Santa Cruz) followed by incubation for 15 min at RT. An equal amount of normal rabbit serum was included as a control. Non-labeled double strand oligonucleotides were used for competition assays. ChIP assays were performed using NSCs transduced with retrovirus expressing either GFP or HA-tagged TLX. NSCs were fixed with 1% formaldehyde for 10 min at RT and the reaction quenched with 0.25 M glycine for 5 min at RT. ChIP was performed as previously described using a rabbit anti-HA antibody (Santa Cruz) (Qin et al., 2013 (link)). Purified DNAs were amplified with the following primer pairs: 5′-TTG TGA CTG ATG GCT ATG GG-3′ and 5′-AAG GGA TTG TGT CTC GCA TAC-3′ for the TLX-binding site and 5′-GGG TTT CAA AGG ATG GTC AAT G-3′ and 5′-AGG CTG GTC TCA AAC TTC TG-3′ for a distal control site.