Criterion 5 tbe precast gels

HepG2 cell nuclear extract with ectopically expressed 3xFLAG-PATZ1 was extracted using NucBuster Protein Extraction kit (Novagen) and quantified with Qubit 2.0 Fluorometer (Life Technologies). The oligonucleotides were minimized to be around 20 bp in length with several nucleotides flanking both sides of the motif sequence for PATZ1. A list of probes used in this assay can be found in Supplementary Table S3. Both biotinylated and non-biotinylated probes were annealed with reverse complementary nucleotide sequences by NEB2 buffer (NEB). A total of 6 μg nuclear extracts were incubated with 200 fmol of biotinylated double-stranded probes for 40 min on ice in 10 mM Tris–HCl, 80 mM KCl, 1 mM DTT, 1 μg of Poly (dA-dT), 7.5% glycerol, 0.063% NP-40 and 2 mM MgCl₂. For competition assay, extra amounts of unlabeled probes were added to the binding reaction and further incubated at room temperature for 20 min. Samples were separated in Criterion 5% TBE Precast Gels (Bio-rad), and electro-transferred into a Genescreen plus hybridization transfer membrane (Perkin Elmer). DNA–protein complexes were cross-linked using UV-light and detected by LightShift Chemiluminescent EMSA Kit (Life Technologies).