Anti nkx 2.5 antibody

ChIP assays were carried out using the Upstate Biotechnology ChIP assay kit as previously described.22 (link) Briefly, chemically cross-linked MCF7 cells were sonicated to yield DNA fragments ranging in size from 200 to 800 base pairs. After centrifugation for 10 minutes at 12,000 rpm, supernatants were recovered, diluted 10-fold into ChIP dilution buffer, and then incubated with 100 μl of 50% protein G-agarose slurry for 30 minutes at 4°C to reduce non-specific interactions between chromatin DNA and protein G-agarose beads. After centrifugation for 5 minutes at 3,000 rpm, supernatants were incubated with 2 μg of anti-Nkx-2.5 antibody (Santa Cruz Biotechnology) or control IgG for 2 hours at 4°C. Immune complexes were then immunoprecipitated by incubation with protein G-agarose beads for 1 hour, and then eluted from the protein G-agarose beads using elution buffer (1% SDS, 0.1 M NaHCO₃). Cross-links were then broken by incubation for 4 hours at 65°C, and DNA was isolated using a PCR purification kit (Qiagen) and used as templates for PCR reactions using primers for MDR1 USPs. Primer sequences are detailed in Table 1.

A promoter enzyme immunoassay was performed as described previously.21 (link) Briefly, biotinylated oligonucleotide probes (5 pmol per well) for wild type or mutated Nkx-2.5-binding site were generated by annealing sense and antisense oligonucleotides and immobilized in streptavidin-coated 96-well plates (Thermo Fisher Scientific) by incubation for 1 hour at room temperature. After washing three times with washing buffer (10 mM HEPES, pH 7.9, 100 mM KCl, 5 mM MgCl₂, 1 mM 1,4-dithiothreitol, 10% glycerol, and 0.5% Nonidet P-40), plates were incubated with nuclear extract (20 µg per well) of MCF7 cells transfected with the plasmid encoding Nkx-2.5 at 4°C for 2 hours in the presence of poly-deoxyinosinic-deoxycytidylic acid (poly-dI/dC; 5 μg per well). Plates were then washed with wash buffer, incubated with anti-Nkx-2.5 antibody (Santa Cruz Biotechnology, 1:1,000) at 4°C for 2 hours, and with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA; 1:2,000) at 4°C for 2 hours. After washing three times with wash buffer, the substrate reagent (R&D Systems, Minneapolis, MN, USA) was added and plates were incubated for 1 hour at 4°C. The colorimetric reaction was stopped by adding 2N H₂SO₄, and absorbance at 450 nm was then measured using a microplate reader (BioRad, Hercules, CA, USA).