Np 40

Electrophoretic mobility shift assay (EMSA) was performed as described with minor modification (Cheng et al., 2008 (link)). Briefly, DNA fragments corresponding to the sequences upstream of putA were amplified by PCR using specific primers (Table S3). DNA fragments (50 ng) were incubated with 2 μg purified rPruR in a 20-μl reaction (50 mM Tris, pH 7.9, 50 mM NaCl, 0.5 mM EDTA, 10% glycerol, 1% [v/v] NP-40 [Solarbio]) at 25°C for 10 min. Samples were loaded onto an 8% native polyacrylamide gel in 1 × Tris-borate-EDTA (TBE) buffer (0.044 M Tris, 0.044 M boric acid, 0.001 M EDTA, pH 8.0) that had been prerun for 1 h, electrophoresed on ice at 100 V for 1.5 h, followed by DNA staining in 1 × TBE containing 0.5 μg/ml ethidium bromide. Bands were visualized with a molecular imager ChemiDoc TM XRS+ (Bio-Rad, CA, USA).

EMSA was performed as previously described with minor modification (Sun et al., 2014 (link)). Briefly, DNA fragments corresponding to sequence upstream of exsA and exsC were synthesized. DNA fragments (200 ng) were incubated with 0 to 6 mM purified recombinant Fis protein at 25°C for 30 min in a 20-μl reaction [10 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 5 mM KCl, 0.1% (v/v) NP-40 (Solarbio), and 1 mM dithiothreitol]. Samples were loaded onto an 8% native polyacrylamide gel in 0.5 × Tris-borate-EDTA (TBE) buffer (0.044 M Tris base, 0.044 M boric acid, 0.001 M EDTA, pH 8.0) that had been prerun for 1 h, electrophoresed on ice at 100 V for 1.5 h, followed by DNA staining in 0.5 × TBE containing 0.5 μg/ml ethidium bromide. Bands were visualized with a molecular imager ChemiDoc™ XRS+ (Bio-Rad).

DH5α cells carrying the pMMB67EH-gst-PA14_51020-His were grown in LB at 37 °C. When the OD₆₀₀ reached approximately 0.6, 1 mM IPTG was added to the medium and the bacteria were kept growing for 4 h at 37 °C. The bacteria were collected by centrifugation at 6000× g for 10 min at 4 °C, followed by resuspension in the lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 3 mM β-mercaptoethanol, 10 mM imidazole, 0.5% NP-40 (Solarbio, Beijing, China), pH 8.0) and sonication. The lysate was centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was incubated with Ni-NTA beads (Qiagen, Düsseldorf, North Rhine-Westphalia, GER) for 2 h at room temperature. The Ni-NTA beads were washed sequentially with the lysis buffer containing 50 mM and 100 mM imidazole. The bound proteins were eluted by the lysis buffer containing 300 mM imidazole. The purity of the eluted protein was examined by SDS-PAGE, and the protein concentrations were quantified by a bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China).

BMSCs of the third passage were taken, and after treatment, when the cells reached 70%–90% confluence, the proteins were extracted. The cells were washed three times with PBS buffer with shaking for 3 min each time, NP40 (Solarbio, Beijing, China) was added to lyse the cells, and the cell lysate was sonicated with an ultrasonic crusher. After centrifugation, the supernatant was removed, 100μL of cell lysate was used as input sample, and the remaining cell lysate was used for immunoprecipitation. Cell lysates were pretreated with Protein A/G agarose beads (Solarbio, Beijing, China), nonspecific binding proteins were removed from lysates, and the supernatant was collected by centrifugation. P53-specific antibodies were added to the supernatant and incubated overnight at 4 °C. Protein A/G agarose beads were pretreated with lysis buffer, Protein A/G agarose beads were added, and samples were incubated for 2 h at 4 °C with rotation. After centrifugation, the supernatant was removed, the immune complexes were washed, loading buffer was added, antigens, antibodies, and agarose beads were dissociated by boiling, and the supernatant was collected by centrifugation. P53 ubiquitination was analyzed by Western blot.

Sequences of the DNA probes are listed in Table S3. DNA fragments (200 ng) were incubated with 0, 2, 4 or 8 μM purified His-tagged PhoP in a 20-μL binding reaction system [50 mM Tris, pH 7.9, 50 mM NaCl, 0.5 mM EDTA, 10% glycerol, 1% (v/v) NP-40 (Solarbio, Beijing, China)] at room temperature for 30 min [37 (link)]. The binding mixtures were loaded onto an 8% native polyacrylamide gel in 1×Tris-borate-EDTA (TBE) buffer (0.044 M Tris, 0.044 M boric acid, 0.001 M EDTA, pH 8.0) that had been pre-run on ice at 100 V for 1 h. The electrophoresis was performed on ice at 100 V for 75 min. The gel was stained with ethidium bromide in 1×TBE buffer for 5–10 min. The bands were visualized with a molecular imager ChemiDoc TM XRS+ (Bio-Rad, Hercules, CA, USA).