Emsa kit

For the yeast one-hybrid assay, the MBS element was synthesized and inserted into the pAbAi vector. The ORF of MYB108A was amplified and fused in-frame with the GAL4 activation domain of the pGADT7 vector. The mutant MBS (mMBS) was used as a negative control. The resultant plasmid was introduced into the yeast strain Y1HGold. The detailed procedure was performed according to the user manual for the Matchmaker Gold Yeast One-Hybrid Library Screening System (Clontech, Mountain View, CA, USA).
For EMSA, the MYB108A ORF was cloned into the expression vector pEASY-E1 (TransGenBiotech, Beijing, China). The MYB108A-His recombinant protein was expressed in Escherichia coli strain BL21 and purified using HIS-tag BeaverBeads™ Nickel (Beaver, BioBAY, China). The ACS1 promoter probe containing an MBS element was synthesized and labeled with biotin (Sangon, Shanghai, China). Unlabeled competitor probes were generated from the dimerized oligos of the ACS1 promoter regions containing the MBS element. EMSA was performed as described in the instruction manual included with the EMSA Kit (Thermo Fisher Scientific, MA, USA).

For the EMSA, a lightshift chemiluminescent EMSA kit (Thermo Scientific, Rockford, IL) was used, and the manufacturer’s protocol was followed. We designed appropriate oligonucleotide pairs that included the putative binding regions for each pump gene. DNA labeling was performed using a Biotin 3´ end DNA labeling kit (Thermo Scientific). The DNA probes were prepared through PCR amplification. DNA (20 fmol) and His-BaeR protein (1 μg) were mixed in binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, and 1 mM DTT) and poly deoxyinosinic-deoxycytidylic acid (poly (dI-dC)) (50 ng/μL). The reaction mixtures (20 μl) were loaded onto a 5% polyacrylamide gel in 0.5 X TBE (45 mM Tris, 45 mM boric acid, and 1 mM EDTA at pH 8.3). The DNA-protein complexes were separated at 100 V for 2 hours in 0.5 X TBE buffer and then transferred to a Biodyne B nylon membrane (Pall Corporation, Port Washington, NY). Crosslinking and detection of the His-labeled DNA-protein complexes were performed using a UV lamp and chemiluminescence, respectively.

Nuclear extract preparation and electrophoretic mobility shift assay (EMSA) were performed using the EMSA kit (Thermofisher, USA) as described previously, with some modifications^{34 (link)}. Briefly, nuclear extract (20 ug) was incubated with binding buffer and nonspecific oligonucleotides for 15 min, and then incubated with NF-κB biotin-labeled oligonucleotide probes (forward: 5′-AGT TGA GGG GAC TTT CCC AGG C-3′; reverse: 5′-G CCT GGG AAA GTC CCC TCA ACT-3′) for another 15 min. For super shift assays, NF-κB antibody was added along with the binding buffer. Subsequently, samples were separated by electrophoresis in a 5.5% polyacrylamide gel with 0.25 × Tris-borate-EDTA buffer. The retarded bands were detected by chemiluminescence.

The CDS sequence of FtbZIP85 was inserted into the protein expression vector pGEX6p-1 and transformed into the Escherichia coli Rosetta (DE3) cells (Novagen, Darmstadt, Germany), which were oscillated and cultured overnight at 37 °C. The prokaryotic expression and purification methods of the protein were described in the instructions for the molecular cloning experiment. The GST-FtbZIP85 (glutathione S-transferase) protein was optimally induced at 16 °C, with an IPTG concentration of 0.6 mM, and the molecular weight of the fusion protein is approximately 34 KDa. Four ABRE motifs were found in the prediction of the FtDFR regulatory regions. Therefore, double-stranded DNA containing four ABRE elements and 15 bases at both ends of the upstream and downstream of FtDFR and double-stranded DNA with mutated motifs were designed and synthesized. The oligonucleotide probes were synthesized. In terms of probe-mBZIP, ABRE was mutated as the negative control. An EMSA kit (Thermo Scientific, China, catalog number: 20148) was used, and detection was carried out using Image Lab 2.0 (Bio-Rad, Hercules, CA, USA). The probes for EMSA are listed in Table S1.

Nuclear Nrf2 DNA binding activity was measured with an EMSA kit (Thermo Fisher), according to the manufacturer’s instructions. Oligonucleotides corresponded to the binding sites of the ARE nucleotide. The oligonucleotide sequences are shown in S1. The specificity of the band was confirmed by competition with cold oligonucleotides and mutated oligonucleotides. Gels were imaged with a scanner (V300, Epson, Japan) and then analyzed with the alphaEaseFC system (Alpha Innotech, Portugal).

Etha promoter DNA with/without biotin-labeling for the DNA-binding activity assays were synthesized by annealing the complementary single-stranded oligo synthesized by Sangon Co., Ltd. (Sangon, Shanghai, China), and then purified by DNA purification Kit (Tiangen, Beijing, China). Ethr (0.43 μM) was incubated with 0, 2, 5, 10, 25, 50, 100, and 200 μM c-di-GMP and 200 μM c-di-AMP and 200 μM cGMP in a total volume of 13 μL PBS buffer at RT for 1 h. Then biotinylated etha promoter was added and co-incubated at RT for 20 min in a total volume of 20 μL EMSA buffer. The reaction mixtures were then subjected to 8% native PAGE, and transferred to a nylon membrane. The membrane was exposed to 254 nm UV light with 120,000 μJ from HL-2000 Hybridization Incubator (UVP, California, US) for 2 min. EMSA assay was completed using EMSA kit (Thermo) according the manufacturer’s instruction. Images were acquired using a Typhoon Scanner (GE Healthcare).