Dig 11 ddutp

We prepared nuclear extracts from rhabdomyosarcoma and A172 cells as described previously [54 (link)]. We prepared probes for the risk (R) and non-risk (N) alleles of rs11190870 by annealing 17-bp complementary oligonucleotides and labeling with digoxigenin (DIG)-11-ddUTP (Roche). For competition experiments, nuclear extracts were pre-incubated with excess unlabeled probes. We detected DNA-protein complexes using a DIG gel shift kit according to the manufacturer’s instructions (Roche).

Electrophoretic mobility shift assays (EMSA) were conducted using a commercial kit (Viagene Biotech, Co., Changzhou, China) following the manufacturer’s protocol. Nuclear extracts were collected from HEK 293T cells as described previously. We prepared probes for the risk allele G and the non-risk allele A of rs1322330 by annealing 21-bp complementary oligonucleotides and labeling with digoxigenin (DIG)-11-ddUTP (Roche, United States). The DNA/protein binding assay was performed with 10 mg of nuclear extracts using the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, United States) according to the manufacturer’s recommendations. For competition experiments, nuclear extracts were pre-incubated with excess unlabeled probes. All gel electrophoresis procedures were performed at 4°C. The DNA/protein complexes were detected by streptavidin peroxidase, and signal detection was performed in a 5200 Multi Luminescent Image Analyzer (Tianneng, China).

We prepared probes for the risk (G) and non-risk (A) alleles of rs1978060 by annealing 25-bp complementary oligonucleotides (sense: 5′-TGTCTAATGTACRCACCAGCTCGGA-3′ and antisense: 5′-TCCGAGCTGGTGYGTACATTAGACA-3′) and labeling with digoxigenin (DIG)-11-ddUTP (Roche). The nuclear extracts were prepared from MCF-7 and FOXA2-overexpressing OUMS-27 cells. DNA–protein binding reactions were performed using a DIG gel shift kit according to the manufacturer’s instructions (Roche). For competition assays, nuclear extracts were pre-incubated with excess unlabeled probes prior to adding DIG-labeled probes. For a super-shift assay, 2 μg of FOXA2 antibody (Santa Cruz; sc-374376X) was added to the reaction mixture and incubated for 20 min at room temperature. DNA-protein complexes were resolved on 6% DNA retardation gels (Thermo Fisher Scientific), and the signal was detected using a chemiluminescent detection system according to the manufacturer’s instructions (Roche). Uncropped gel images can be found in the Source Data file.