Gelshift chemiluminescent emsa kit

Electrophoretic mobility shift assay was performed according to a previous study. 25 Briefly, oligonucleotide probes with a biotin tag at the 5'‐ end of the sequence (Integrated DNA Technologies) were incubated with HEK293T nuclear protein and the working reagent from the Gel shift Chemi‐luminescent EMSA kit (Active Motif 37341). The wild‐type E2F3 EMSA probe sequences were 5'‐GGA ATA CTA ATA AGT CTT AAA AGT TC‐3' and the mutant E2F3 EMSA probe sequences were 5'‐GGA ATC TGC CAA GTC TGC CCA GTT C‐3'. For competitor assays, an unlabelled probe was added to the reaction mixture at 100× excess. The reaction was then incubated for 30 minutes at room temperature and then loaded onto a 6% retardation gel (Thermo Fisher Scientific EC6365BOX) and run in 0.5× TBE buffer. After transfer onto a nylon membrane, the membrane was visualized with the chemiluminescent reagent as recommended. The super shift assay was performed with 1 μg anti‐QKI‐6 antibody (Millipore) and incubated on ice with protein from HEK 293T for 30 minutes prior to addition of oligonucleotide probes and gel electrophoresis.

Protein-DNA binding was assayed with the Gelshift Chemiluminescent EMSA kit (Active Motif) following the manufacturer’s protocol. For the SOX9 protein, the binding reactions were carried out in 20 μL of binding buffer containing 1 μg of poly(dG-dC)·poly(dG-dC) and 20 μg of BSA. To generate the DNA probes or competitors above 100 bp in size, double-stranded DNA (dsDNA) fragments were amplified by PCR with the biotin-labelled or unlabeled PCR primers listed in Supplementary Table 2, respectively. The biotin-labelled DNA probes less than 100 bp in size were prepared from synthesized DNA oligonucleotides using the Biotin 3′ End DNA Labeling kit (Thermo Fisher Scientific). Nuclear proteins were isolated using the Nuclear Extract Kit (Active Motif) from HEK293T cells transfected with pcDNA3 vectors to express FLAG-PAX1, FLAG-PAX9, and SOX9 protein or pCAG-IRES-EGFP vectors to express PAX1 and SOX9 protein. Five micrograms of anti-FLAG M2 monoclonal antibody (Sigma, F3165) was used for each reaction of the antibody with the nuclear protein.

EMSA probes were synthesized by Integrated DNA Technologies. The Gelshift Chemiluminescent EMSA kit (Active Motif, Carlsbad, CA) was used according to the manufacturer's protocols. Baculovirus recombinant human GR protein (ab3582; 3.5 μg) for each condition were incubated with probes (20 fmol) (Supplementary Table 3) for 20 min on ice. For supershift, anti-GR (ab3578) from Abcam (Cambridge, MA) was incubated for 30 min after adding probes. The 5% TBE gel was run at 100 V for 1 h and then transferred to nylon membrane. After incubation with streptavidin–horseradish peroxidase conjugate, the membrane was exposed to film and developed.

Gel shift assay was conducted using a double-stranded, biotin-labeled oligonucleotide probe containing the consensus binding site for STAT5 (sense strand, 5′-AGATTTCTAGGAATTCAATCC-3′), using the Gelshift Chemiluminescent EMSA kit (Active Motif). In brief, protein–DNA complexes were resolved on a nondenaturing polyacrylamide gel, transferred to a positively charged nylon membrane, and cross-linked to the membrane using the UV-light cross-linker. After blocking, the membrane was incubated with blocking buffer containing streptavidin conjugated to HRP. After washing, protein–DNA complexes were detected using a chemiluminescent substrate (Active Motif, Carlsbad, CA, USA)^{40 (link),49 (link)}.

GBM8 glioma patient-derived cells were treated with Olig2 inhibitor at 5 μM or DMSO vehicle control for 20 hrs. Nuclear and cytoplasmic fractions were then isolated using ActiveMotif Nuclear Extraction Kit (Cat No. 40010). Nuclear extracts were used for electrophoretic mobility shift assay (EMSA). To detect Olig2 binding DNA the Gelshift Chemiluminescent EMSA Kit from ActiveMotif was used (Cat No. 37341). The 27 bp oligonucleotide sequence (sense strand 5′-gctcagagcccagctgctggactgagc -3′) with Olig2 binding site (bold and underlined) was synthesized by IDT and contained a biotin tag at the 5′terminus of both strands. EMSA was performed according to kit instructions; nuclear extract (5 ug of protein) was incubated for 20 minutes at room temperature with 20 fmol biotinylated DNA. Nuclear and cytoplasmic extracts were subjected to immunoblot analysis with antibodies to Olig2 (Dana-Farber Ab#308), PhosphoSerine 10,13,14-Olig2 (Abcam), Lamin A/C (ActiveMotif), E47 (E2A) (Cell Signaling Technology), Tubulin (Sigma).