Nylon membrane

Southern blot was performed as described before
⁶⁶ (link). In brief, cells containing KmYACs were embedded in agarose plugs. Spheroplasts were created and lysed in the plugs. Subsequently, the plugs were subjected to pulsed‐field electrophoresis (Bio‐Rad). Linear KmYACs were resolved directly. Circular KmYACs were entrapped in the agarose plugs and separated from the natural chromosomes during electrophoresis. Plugs containing circular KmYACs were digested with NotI and subjected to the pulsed‐field electrophoresis again to release the linearized KmYACs. KmYACs were transferred from gel to a nylon membrane (Beyotime) for about 16–20 h. The membrane was hybridized with DNA probes prepared using a Biotin Random Primer DNA Labeling Kit (Beyotime) and then detected using Chemiluminescent Biotin‐labeled Nucleic Acid Detection Kit (Beyotime). The probe targets HphMX4 and its sequence is listed in Table S1.

Recombinant p‐GST‐bZIP72 (phosphorylated GST‐bZIP72) was purified from Transetta (DE3) chemically competent cell (Transgen) coexpressing GST‐ bZIP72 and His‐SAPK10. The EMSA probes in a length of 59 nt in the AOC promoter were commercially synthesized by Tsingke Co. (Tsingke Biotech, Hangzhou, China) and labeled using an EMSA Probe Biotin Labeling Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Probes used were listed in Table S1. An equal amount of purified recombinant proteins were preincubated with EMSA/gel‐shift binding buffer (Beyotime) at 25°C for 20 min, then incubated with 20 fmol labeled probes with or without nonlabeled competitive DNA probes for another 20 min. Then the incubated mixed samples were separated by electrophoresis on 6% acrylamide gels and transferred to the Nylon membrane (Beyotime, Shanghai, China). The labeled DNA probes was detected using the LightShift Chemiluminescent EMSA kit (Thermo) according to the manufacturer’s instructions. Migration of biotin‐labeled probes was detected using Supersignal West Pico Chemiluminescent Substrate (Thermo) and the ChemDoc™ Touch Imaging system (Bio‐Rad).

Purified MBNL1 protein (CUSABIO), cytoplasmic or nuclear protein separated from MDA‐MB‐453, was incubated with tRF‐1‐Ser biotin‐labelled (RIBOBIO), tRF‐1‐Ser biotin‐unlabelled (RIBOBIO), MBNL1 antibody (Thermo Fisher) or tRF‐1‐Ser‐UGCU‐mutation biotin‐labelled (RIBOBIO) in REMSA Binding Buffer (Beyotime) at 4°C for 10 min. The RNA–protein complex was separated with a 6% Tris‐Borate‐EDTA buffer (TBE) PAGE gel in ice box, then transferred to a nylon membrane (Beyotime). The membrane was then incubated with streptavidin–horseradish peroxidase (HRP) conjugate at 20°C for at least 20 min. The signal of the RNA–protein complex was detected using ChemiDocXRS (BIO‐RAD).

The His6-CpxR protein was expressed using E. coli BL21 (DE3)-containing pET30a-CpxR and purified by using a Ni-nitrilotriacetic acid (Ni-NTA) resin affinity chromatography (Qiagen, Germany). The purified protein was phosphorylated by acetyl phosphate (Sigma, United States) according to previously described procedures (Li et al., 2018 (link)). DNA probes were amplified and purified and then labeled using a Biotin Labeling Kit (Beyotime, China). Then, the phosphorylated CpxR and labeled probes were used for protein-DNA EMSAs as described previously (Cheng et al., 2021 (link)). Labeled DNA probe (1 μM) and various concentrations of phosphorylated CpxR protein (0–4 pmol) were incubated at 24°C for 20 min in reaction buffer (50 mM Tris–HCl, pH 8.0, 2.5 mM MgCl₂, 100 mM KCl, 0.2 mM DTT, 10% glycerol, 2 μg salmon sperm DNA). For competition experiments with unlabeled DNA probes, a 100-fold molar excess was preincubated with phosphorylated CpxR protein. The reaction mixtures were loaded on a 4% non-denaturing polyacrylamide electrophoresis in 0.5 × Tris-borate-EDTA (TBE) buffer. The bands of labeled probes were subsequently transferred to nylon membrane (Beyotime, China), and detected using the Chemiluminescent EMSA Kit (Beyotime, China).

The expression vector pET-28a-cpxR was constructed, and the recombinant protein CpxR was obtained and phosphorylated according to a previously reported procedure (12 (link)). The promoter regions of the apfA and pilM genes were amplified by PCR and labeled by terminal deoxynucleotidyl transferase (TdT) using an EMSA probe biotin labeling kit (Beyotime, China), generating biotin-labeled probes. The binding of His-CpxR to the biotin-labeled probes was performed using a chemiluminescent EMSA kit (Beyotime) according to the procedure recommended by the manufacturer. Samples were loaded onto a 4% nondenaturing polyacrylamide gel buffered with 0.5× Tris-borate-EDTA (TBE) and blotted onto nylon membrane (Beyotime). Visualization was performed using the chemiluminescent EMSA kit (Beyotime).

Total RNAs were denatured at 65°C for 5 min. RNA (300 ng) was spotted onto a nylon membrane (Beyotime). The membranes were UV-crosslinked for 10 min, then blocked with 5% milk, and incubated with anti-m⁶A antibody. After washing with TBS-T, the membranes were probed with the corresponding HRP-conjugated secondary antibodies. The blots were visualized using the ECL system (Thermo Fisher Scientific). The membranes were also stained with 0.02% methylene blue (Sangon Biotech, Shanghai, China) in 0.3 M sodium acetate. methylene blue staining was used for RNA loading control.