Electrophoretic Mobility Shift Assay

EMSA was performed as previously described with minor modifications (Hellman and Fried, 2007 (link)). We used NCTC8325 as the template to amplify katA promoter region DNA fragment. The 30 ng DNA fragment was incubated with 0, 100, 200, and 400 ng purified ArcR in binding buffer (25 mM HEPES, 1 mM dithiothreitol, 200 mM NaCl, and 10% glycerol, pH 7.8) at 37°C for 30 min. The 8% polyacrylamide gel was pre-electrophoresed in 1× Tris-borate-EDTA buffer (0.044 M Tris, 0.044 M boric acid, and 0.001 M EDTA, pH 8.0) for 1 h to remove impurities. After adding the sample, electrophoresis was performed for 1 h 40 min on ice. At the end of the electrophoresis, the glue was stained with 0.5 μg/ml ethidium bromide. Imaging was performed using a gel imager (Bio-Rad, Hercules, CA, USA).

For generation of the labeled probe, 5ʹ-IRDye® 700 labeled oligonucleotides were purchased from IDT with the following sequences: ABE; 5ʹ-CGG TGT TGC ACG CGG *CGG GAC GCT CGC GGT AGT TTT* TTC CCA TGA TCA CG-3ʹ and 5ʹ-CGT GAT CAT GGG AAA *AAA CTA CCG CGA GCG TCC CGC CGC* GTG CAA CAC CG-3ʹ and scrambled control probes; 5ʹGTT TAC TAG GTC GAG GTA CTT CGA CGC GCG CCG TCT GCT AGC GCG GTC TG-3ʹ and 5ʹ-CA GAC CGC GCT AGC AGA CGG CGC GCG TCG AAG TAC CTC GAC CTA GTA AAC3ʹ. The AlpA binding element is indicated by asterisks. The oligonucleotides were annealed by mixing them in equimolar amounts in duplexing buffer (100 mM Potassium Acetate; 30 mM HEPES, pH 7.5) and heating to 100 °C for 5 min in a PCR cycler. The cycler was then turned off and the samples were allowed to cool to room temperature while still inside the block. The annealed product was then diluted with water to 6.25 nM for EMSA experiments.
For EMSAs fluorophore labeled DNA probes at a concentration of 0.3125 nM were incubated for 30 min at 20 °C in 20 µl reaction mix (Licor Odysee EMSA Kit) containing 33.4 mM Tris, 70.2 mM NaCl, 12.5 mM NaOAc, 3.75 mM HEPES, 50 mM KCl, 3.5 mM DTT, 0.25% Tween20 and 0.5 µg sheared salmon sperm DNA (ThermoFisher) with proteins. For resolving the reactions, 4% polyacrylamide gels containing 30% triethylene glycol were cast (For two gels: 2 ml ROTIPHORESE®Gel 30 37.5:1 (Roth), 4.5 ml triethylene glycol (Sigma-Aldrich), 1.5 ml 5x TBE-buffer, 7 ml ddH2O, 15 µl TEMED, 75 µl 10% APS). The gels were preequilibrated for 30 min at 130 V in 0.5x TBE-buffer. Samples with added 10x orange dye were then loaded onto the gel at 4 °C and the voltage set to 300 V until the samples entered the gel completely. The voltage was then turned down to 130 V and the gel was run until the migration front reached the end of the gel. The gels were imaged using the Licor Odyssey imaging system using the 700 nm channel. For generation of the figures, the scanned image was converted to greyscale and brightness and contrast adjusted. The unprocessed scan is available as Supplementary Fig. 15.

A total of 29 base pairs sense (S) and antisense (A) oligonucleotides encompassing the rs72705342:C>T (NC_000014.9:g.91890855C>T) were synthesized for performing the EMSAs (Supplementary Table S2). The oligos were synthesized with their 5′-end labelled with biotin and unlabelled oligos were procured as well. The oligos were annealed by incubating the mix of complementary strands at 95°C for 5 min followed by gradually cooling down the mix to room temperature. Nuclear extract from HLE B-3 cells was prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific, U.S.A.). The binding reaction included poly (dI.dC) as non-specific competitor DNA. For competition experiments, a 200-fold excess of unlabelled oligonucleotides was included in the pre-incubation mixture. For supershift assays, EMSA-specific antibodies for TFII I (sc-46670X, Santa Cruz, U.S.A.) and GR-α (PA1516, Invitrogen) were pre-incubated with the nuclear extract for 1 h before adding the final reaction mixture. The complexes after incubation were resolved on 6% native polyacrylamide gels and transferred to nylon membranes and developed. The EMSA was performed with the Lightshift Chemiluminescent EMSA kit (Thermo Fisher Scientific, U.S.A.). Detection was done using Fusion Solo S Chemi-Doc (Vilber Lourmat) and gel shifts were quantified with the Evolution Capt software (Vilber Lourmat Fusion Solo S).