Las600

Binding of proteins onto DNA probes was determined with the electrophoretic mobility shift assay (EMSA) as described previously (Lv et al., 2018 (link)). PhcA and PhcR proteins were purified from the pET-32a based protein expression system in E. coli BL21 (DE3) and subjected for the binding reaction with biotinylated DNA probes using the LightShift Chemiluminescent EMSA Kit following the manufacturer’s protocol (Thermo, United States). Promoter regions of the rmyA operon (rmyA upstream region of ATG site about 375 bp) and impH gene (424 bp of its coding sequence) were PCR amplified and labeled by biotin using the Biotin 3′ End DNA Labeling Kit (Thermo, United States). Note that promoter DNA of impH gene, which encodes one of the type VI secretion proteins in R. solanacearum, was selected as a system control. The gels were stained by SYBR GOLD dye and subjected to screening on a phosphor screen (LAS600, GE Healthcare).

Cells were lysed with Laemmli sample buffer containing 2.5% β-mercaptoethanol and heated at 95°C for 5  min. Samples were loaded to 4–15% polyacrylamide gels (Bio-Rad) for electrophoresis. Proteins were then transferred to a Poly Vinylidene DiFluoride (PVDF) membrane (Merck), which was blocked with 5% dry milk, Tris buffered saline, 0.2% Tween, and incubated with primary antibodies (overnight at 4°C) followed by secondary antibodies (1:10000) for 1  hr at room temperature. Proteins were detected by using Luminata Crescendo (Merck) and LAS600 (GE Healthcare). The following antibodies were used: anti-MMP14 rabbit monoclonal (1:1000, EP1264Y, Abcam), anti-alpha-catenin rabbit monoclonal (1:1000, EP1793Y, Abcam), anti-Vimentin mouse monoclonal (1:1000, 1A4, Sigma), anti-Fibronectin rabbit polyclonal (1:1000, Sigma), anti-integrin β1 mouse monoclonal (1:1000, P5D2, Abcam), anti-integrin β3 rabbit monoclonal (1:1000, ERP17507, Abcam), and anti-actin mouse monoclonal antibody (1:2000, AC-40, Sigma).

Various amounts of Dnr proteins were mixed with different DNA promoters in 20 μL of the gel shift loading buffer (25 mM Tris-HCI [pH 7.5], 10 mM MgCl, 1 mM DTT, 10% glycerol, 15 μg/mL sheared salmon sperm DNA). After incubation at room temperature for 15 min, aliquots of the binding reaction mixtures were then electrophoresed on 7% nondenaturing acrylamide gels in 0.5× Tris-borate-EDTA (TBE) buffer. The gels were stained by Gelstain and subjected to screening on a phosphor screen (LAS600; GE Healthcare).