Orange loading dye

Human purified His-tagged NRF2 (75 nM), MAFG (37.5 nM), FOS (75 nM), JUN (75 nM), or TNT- prepared human NRF1 (2μl/20μl reaction), and IRDye-700 labeled double stranded DNA were incubated for 30 min at room temperature with poly(dI-dC) in the binding buffer (20 mM HEPES, 4 mM MgCl₂, 100 μg/ml BSA, 4% glycerol, 20 mM KCl, 5 mM DTT, and 1 mM EDTA). Orange loading dye (LI-COR, Lincoln, NE) was added and samples were electrophoresed through a native acrylamide gel in 1X TBE. Gels were imaged using the Odyssey infrared imaging system (LI-COR, Lincoln, NE). Double stranded oligonucleotides containing the following sequences were used EMSA: 5’-AATCCGCAGTCACAGTGACTCAGCAGAATCTGAGCCTAG-3’ (wild-type NQO1 ARE, NQO1^WT); 5'-AATCCGCAGTCACAGACTCCTACGAGAATCTGAGCCTAG-3’ (mutant NQO1 ARE, NQO1^mut), 5’-CTGCGCTCCAAACCCGCTGTGTCATACCATACCGGATGT-3’ (MS4A6A ARE, rs667897^A allele), 5’-CTGCGCTCCAAACCCGCTGTGTCGTACCATACCGGATGT-3’ (MS4A6A ARE, rs667897^G allele).

EMSA for IRP/IRE binding was performed based on the published protocol (48 ) with the following modification. The cytosolic fractions were prepared from eWAT, iWAT, and iBAT. Two micrograms of cytosolic protein was incubated with 16 μl of reaction mixture containing 5% glycerol, 0.2 units Super RNasin (Promega, N2511), 2 μg of yeast tRNA (Thermo Fisher, AM7119), 50 uM DTT, and 50 nM of IRDye700-IRE consensus RNA oligonucleotides in 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, and 100 mM KCl for 30 min at room temperature. The resulting reaction mixture was mixed with 2 μl of Orange loading dye (Li-Cor, P/N 927-10100), and load on a 4 to 12% acrylamide/TBE gel (Invitrogen) and run at 120 V for 3 h in the dark. The gel was scanned using Odyssey CLX System (Li-Cor). The RNA oligonucleotide consensus to IRE labeled with IRDye700 probe was synthesized from Integrated DNA Technology based on the published sequence; 5′-UCCUGCUUCAACAGUGCUUGGACGGAAC-3′ (49 (link)).

Nuclear proteins were isolated using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, USA) according to the manufacturer's instructions. A protease inhibitor cocktail (Roche Molecular Biochemicals; Mannheim, Germany) was added to all of the samples, and the BCA protein assay reagent kit (Pierce; Rockford, USA) was used to quantify nuclear proteins. All samples were stored at −80°C. EMSA was performed using an Odyssey Infrared Imaging System with NF-κB IRDye-labelled oligonucleotides (LICOR Inc., Lincoln, USA). The DNA-binding reaction used 10 mg of nuclear extract mixed with oligonucleotide and gel shift-binding buffer (Tris-HCl 100 mM, pH 7.5, KCl 100 mM, DTT 2.5 mM, 0.25% Tween-20, MgCl2 5 mM, 20% glycerol, EDTA 2.5 mM, and polydeoxyinosinic-polydeoxycytidylic acid 1 mg/ml). Reactions occurred at room temperature in the dark for 30 minutes. Two microliters of orange loading dye (LICOR Inc., Lincoln, USA) was added and sample-loaded on the prerun 6% polyacrylamide gels. NF-κB p65 antibody (1 : 1000, Cell Signaling Technology, Danvers, USA) and unlabelled NF-κB oligonucleotides were used to confirm the supershift and specificity of NF-κB DNA-binding activity. The gels were scanned, and the signals were quantified using an Odyssey Infrared Imaging System and Odyssey software (LICOR Inc., Lincoln, USA).

All EMSAs were performed with SpCas9-NLS protein (CAS9PROT from Sigma-Aldrich) in EMSA buffer (20 mM Tris-HCL pH 7.4, 150 mM KCl, 5 mM MgCl₂, 0.1% BSA, 1 mM DTT, 5 mM EDTA, 200 U/mL Superase-In RNase Inhibitor (ThermoFisher Scientific), 5% glycerol, 0.01% Tween 20, 50 µg/mL heparin). RNA was in vitro transcribed with the MEGAscript T7 Transcription kit (ThermoFisher Scientific) and purified with RNA Clean & Concentrator-5 (Zymo). Labeled RNA was 5′ labeled with the 5′ EndTag Labeling DNA/RNA kit (Vector Laboratories) and IRDye 800CW Maleimide (Li-COR Biosciences). After incubating protein and RNA in EMSA buffer for 30 min at room temperature, 10x Orange loading dye (Li-COR Biosciences) was added to samples before pipetting into gels pre-run for 20 min at 120 volts at 4 °C. Gels were resolved by running for 1 h at 120 volts at 4 °C on 6% Novex TBE gels (ThermoFisher Scientific) with 0.5x TBE buffer. Images were taken with the Azure Biosystems c600 imager.

Full-length Nus-His-FAN1 proteins were expressed in E. coli and partially purified in one step using cobalt agarose^{11 (link)}. Further purification of FAN1 proteins was attempted, but active protein yields were low. Proteins were stored in 50 mM Tris-Cl pH 7.5, 270 mM sucrose,150 mM NaCl, 0.1 mM EGTA, 0.03% Brij-35 and 0.1% β-mercaptoethanol.
Double-stranded DNA substrate containing a 5′ single-strand overhang with an IR-700 fluorescent label (5′ flap DNA) was generated by annealing three oligonucleotides (TOM112, TOM117 and TOM122, based on published sequences^{64 (link)}) at 95 °C for 10 minutes and cooling slowly to room temperature overnight. Nuclease assays were carried out by pre-incubating 10 nM Nus-His-FAN1 protein, normalized using FAN1 band intensities from SDS-PAGE (Supplementary Fig. 3b), with 5 nM 5′ flap DNA in 9 μl of reaction buffer (25 mM Tris-Cl pH 7.4, 25 mM NaCl, 0.1 mg ml⁻¹ of BSA and 0.1 mM DTT) for 5 minutes on ice and then adding 1 μl of MnCl₂ (10 mM) and incubating at 37 °C to start reactions. Reactions were continued for 4 minutes before quenching with 10 μl of stop buffer (1× TBE containing 12% Ficoll Type 400, 7 M urea, orange loading dye (LI-COR Biosciences) and 4 mM EDTA). Samples were denatured at 95 °C for 5 minutes and run on a 15% TBE urea gel (200 V, 1 hour) before imaging and quantitation on an Odyssey CLx (LI-COR Biosciences).