EMSA was carried out using a LightShift Chemiluminescent EMSA kit (Thermo Scientific) as described previously. Briefly, the double-stranded 5′-biotinylated DNAs were synthesized as Table S5 . After incubating with probes for 20 min, the nuclear extracts from 661W were separated by 7% EMSA gel and transferred onto Biodyne B precut modified nylon membranes (Thermo Scientific). The membranes were cross-linked in a UV transilluminator for 15 min and then were incubated with blocking buffer and streptavidin-horseradish peroxidase conjugates. Bound conjugates were detected using a molecular imager (Chemi Doc XRS+, Bio-Rad).
Biodyne b precut modified nylon membranes
Manufactured by Thermo Fisher Scientific
Biodyne B precut modified nylon membranes are a type of laboratory equipment used for various applications. These membranes are made of nylon and have been pre-cut to a specific size. The core function of these membranes is to provide a surface for the immobilization and analysis of biomolecules, such as proteins, nucleic acids, or other materials.
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3 protocols using biodyne b precut modified nylon membranes
1
Electrophoretic Mobility Shift Assay
2
Electrophoretic Mobility Shift Assay of HIF-1α and HRE Binding
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The binding activity of HIF-1α and HRE on the MUC5AC promoter was detected by electrophoretic mobility shift assay (EMSA). The single-stranded oligonucleotide sequence of 5′-ccc acc cac gtg aag cac g-3′ , 3′-cgt gct tca cgt g gg tgg g-5′ , which corresponds to the HIF-1α binding site, was end labeled with biotin (100 pmol; Bioneer, Daejeon, Korea). Cells were resuspended in cell homogenization buffer containing 0.05% (v/v) nonidet P-40 and homogenized. Next, nuclei were pelleted and resuspended in cell resuspension buffer (40 mM HEPES at pH 7.9, 0.4 M KCl, 1 mM dithiothreitol, 10% v/v glycerol, 0.1 mM phenlmethylsulfonylfluoride, 0.1% w/v aprotinin, and 0.3 M NaCl). The nuclear extract was centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatant was aliquoted and stored at −70°C. Nuclear extract (5 µg) was incubated at room temperature for 20 min with biotin-labeled HRE oligonucleotide in Chemiluminescent Nucleic Acid Detection Kit (Thermo; Rockford, IL, USA). The oligo-nuclear-extract complex was separated by electrophoresis through 6% (w/v) nondenaturing polyacrylamide gels in 0.5× Tris borate-EDTA buffer. The gel was transferred to Biodyne B precut modified nylon membranes (Thermo). Blotting was done with the Chemiluminescent Nucleic Acid Detection Kit (Thermo).
3
EMSA Analysis of AP1 Transcription Factor
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The EMSA kit was obtained from Pierce (Thermo Fisher Scientific, Inc.). Cells were harvested for nuclear extraction using a nuclear and cytoplasmic extraction kit (Thermo Fisher Scientific, Inc.). The probe sequences were as follows: AP1: 5'-GAC TGG TTG ACT AAG TCA AT-3'. Mut AP1: 5'-GAC TGG CCT ACC GGG TCA AT-3'. Biotin-5'-end-labeled and unlabeled, sense and antisense oligonucleotides were synthesized. The oligonucleotides were annealed to generate double-stranded probes. EMSA was performed by preincubating 3 µg nuclear extract with a mixture containing 1 µg poly dI:dC and 2 µl binding buffer on ice for 10 min. Then, 20 fmol biotin-labeled double-stranded probe was added to the mixture and incubated at room temperature for another 30 min. Competitive EMSA experiments were conducted by incorporating excess concentrations of the unlabeled probe in the pre-incubation step of the assay. DNA-protein complexes were resolved on a non-denaturing 5% polyacrylamide gel in 0.5X Tris-borate-EDTA. After electrophoresis, the samples were transferred onto Biodyne B precut modified nylon membranes (Thermo Fisher Scientific, Inc.). The membranes were cross-linked in a UV transilluminator for 15 min and then were incubated with blocking buffer and streptavidin-horseradish peroxidase conjugates. Bound conjugates were detected using a molecular imager (ChemiDoc XRS+; Bio-Rad Laboratories).
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