Positively charged nylon membrane

In order to determine the relative affinity of ZNF32 for double-stranded DNA, the 5′-biotin-labeled oligonucleotide 5′-tttgtagtcaagtgcattttagccacaaagat-32, corresponding to the human SOX2 promoter, was synthesized by Invitrogen. The GST-ZNF32 fusion protein was purified as described above. Nuclear proteins from BE(2)-C stable cells were prepared using the ProteoJET™ Cytoplasmic and Nuclear protein Extraction Kit (Thermo Scientific) according to the manufacturer's instructions. Approximately 10 μg of nuclear protein or 2 μg GST-ZNF32 fusion protein was incubated with 2 nM biotin-labelled double-stranded oligonucleotide probe in reaction buffer for 20 min at room temperature using the LightShift™ Chemiluminescent EMSA Kit (Pierce Biotechnology). Samples were subjected to 5% nondenaturing gel electrophoresis in 0.5×Tris-borate EDTA, transferred to a positively charged nylon membrane (Millipore), and the membrane was developed using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific) according to the manufacturer's instructions. For competition assays, a 200-fold excess of unlabeled probe was included to the binding reactions.

As described in a previous study, the 11 E. coli strains were subjected to S1-PFGE to identify the number and size of plasmids [28 (link),29 (link)]. The overnight cultures were washed with PBS buffer and then embedded in agarose plugs. The plugs were digested with proteinase K at 37 °C, shaking at 120 rpm for 2 h, and were restricted with S1 nuclease. Salmonella H9812 was restricted with XbaI, which was used as the size marker. A Southern blot was used to confirm the positioning of the mcr-1 or bla_NDM genes on the plasmids. The DNA fragments were transferred to a positively charged nylon membrane (Millipore, USA) by wet transfer and then hybridized according to the protocol as the DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche, Germany).

Filter-mating conjugation experiments were performed between 22 different isolates. E. coli J53 resistant to sodium azide was used as the recipient strain. Transconjugants that possessed the bla_NDM-5-bearing plasmid were selected on Mueller-Hinton agar (MHA; Oxoid) plates that contained 180 μg/ml sodium azide with 1 μg/ml meropenem. Antimicrobial susceptibility testing and PCR amplification of the transconjugants were subsequently performed to confirm whether the plasmid was successfully transferred to the recipient. The PBRT 2.0 kit for PCR-based replicon typing was used for molecular typing of plasmids (Diatheva, Fano, Italy). Plasmid relationships were tested by restriction fragment length polymorphism (RFLP) using HindIII and EcoRI. Digested plasmid DNA was electrophoresed in a 0.8% agarose gel for approximately 1 h. Four strains were selected for further study. Plasmid stability was tested by liquid experiments as previously described (34 (link)). S1-PFGE and Southern blotting were further performed to determine the plasmid location of the bla_NDM-5 gene. Genomic DNA digested with S1 nuclease was subjected to PFGE as described above. The DNA fragments were transferred to a positively charged nylon membrane (Millipore, USA) and then hybridized with a digoxigenin-labeled NDM-5-specific probe. S. enterica serotype Braenderup H9812 was used as the size marker.