Chemiluminescent nucleic acid detection kit

Northern blots were performed using a NorthernMax kit (Ambion) according to the manufacturer’s instructions. Briefly, 1.5 μg of total RNA for each strain of S. aureus were separated on a 1% MOPS (morpholinepropanesulfonic acid)-formaldehyde-agarose gel and transferred to a BrightStar-Plus positively charged nylon membrane (Invitrogen) using a Whatman Nytran SuPerCharge TurboBlotter kit (GE Healthcare Life Sciences) for 3.5 h. Samples were cross-linked to the membrane by baking at 80°C for 20 min. Biotin-labeled RNA probes were synthesized from DNA with gene-T7-specific primer sets (see Table S1 in the supplemental material) using a MaxiScript T7 transcription kit (Thermo Fisher), including the optional DNase digestion and cleanup with NucAway spin columns (Invitrogen). Probes were added to 10 ng/ml in Ultrahyb ultrasensitive hybridization buffer (Invitrogen), followed by incubation at 72°C for 16 h. The membranes were washed as directed using a NorthernMax kit, with the two high-stringency washes performed at 68°C. RNA was visualized with a chemiluminescent nucleic acid detection kit (Thermo Fisher) according to the manufacturer’s instructions.

Total RNAs from yeast cells were extracted using a hot-phenol method. Overnight cultures were treated at 37°C for 2 h, pelleted, and resuspended in 0.3 M sodium acetate pH 4.5. One volume of acidic phenol was then added. The samples were vortexed for 10 s and incubated on ice for 15 min with occasional vortexing. The aqueous phase was collected after centrifugation at 13 000 rpm for 15 min at 4°C. Two volumes of ice-cold 100% ethanol were added, and the samples were kept at −80°C for 1 h. Pellets were collected by centrifugation for 20 min at 13 000 rpm, washed with 70% ethanol, dried at room temperature for 5 min, and dissolved in sodium acetate buffer (pH 5.2). Total RNAs were separated on an acidic urea gel prepared with 0.1 M sodium acetate buffer pH 5.2. Deacylated tRNA^Thr control was obtained by treating the RNA samples in Tris buffer pH 9.0 for 1 h at 42°C. For northern blot, the nylon membrane was treated with Ultrahyb™ hybridization buffer (Invitrogen) at 42°C for 1 h after UV crosslinking, and incubated with 100 ng/μl biotin-labeled DNA probe (Biotin-TTGAACCGATGATCTCCACA) at 42°C overnight. Finally, tRNA^Thr and aa-tRNA^Thr were detected using the Chemiluminescent Nucleic Acid Detection Kit (Thermo Fisher Scientific).

Nuclear proteins were prepared as described previously [22 (link)]. EMSAs were performed by using a Lightshift chemiluminescent EMSA kit according manufacturer’s instruction (Thermoscientific, IL, USA). The biotin-upper and biotin-lower strand of Stat3 probe (5′-cttcatttcccggaaatcccta-Biotin-3′ and 5′-tagggatttccgggaaatgaag-Biotin3′) were used. The biotin-labeled DNA probes were incubated with PAA-treated nuclear proteins in a final volume of 20 μL EMSA buffer containing 1 μg/μL poly (dI-dC) at room temperature for 20 min. The protein/DNA complexes were run on a 4% polyacrylamide nondenaturing gel in 0.5× TBE (45 mM tris-borate and 1 mM EDTA) and detected using a chemiluminescent nucleic acid detection kit (Thermo Scientific).

Single-stranded complementary oligonucleotides containing predicted CSL binding sites were labeled using a Biotin 3′ end DNA labeling kit (Thermo Scientific, 89818). Double-stranded DNA probes were made by annealing biotin-labeled complementary oligonucleotide pairs at room temperature for one hour. 0.5 μM purified mouse Rbpj protein (residues 53–474; Friedmann et al., 2008) and 1 nM of labeled oligonucleotide complexes, in the presence of 1.9 ng/μl poly[d(I-C)] (Sigma, 10108812001), were resolved on a 6% DNA retardation gels (Invitrogen, EC63652BOX) in 0.5 × TBE buffer and then transferred to nylon membranes. The LightShift Chemiluminescent EMSA kit assay (Thermo Scientific, 20148) was performed on the blots, which were developed using the Chemiluminescent Nucleic Acid Detection kit (Thermo Scientific, 89880), Kodak x-ray film and film developer.

We mixed 1–5 µl of MBP-hATx-6 at 9.2 μg/μl, 1.0 µM of TE end(s) and 50 ng of supercoiled pBR322 DNA in a 20 µl reaction containing 25 mM MOPS pH 7.0, 0.2 mM TCEP, 5 mM MgCl₂, 50 μg/ml BSA, 100 mM NaCl, 0.1 mM EDTA and incubated at 30°C for 30 min. We stopped the reaction with 5 µl of 6x Purple Gel Loading Dye (NEB) and loaded 15 µl on a 1% agarose gel containing 0.5 μg/ml ethidium bromide. We measured activity by conversion of the supercoiled plasmid to linear and nicked forms. For assays where the TE ends were labelled with biotin, we followed transfer of the end to the plasmid DNA by electroblotting the DNA in the gel to a positively charged nylon membrane (Sigma # 11417240001) using a Bio-Rad Trans-Blot apparatus at 200 mA for 30 min. We washed and labelled the membrane using the Chemiluminescent Nucleic Acid Detection kit (ThermoFisher #89880) and visualized on a Amersham Typhoon Gel Imaging scanner (GE Life Sciences) with 488 nm laser and the Cy2 filter.

Electrophoretic mobility shift assays (EMSA) employed biotin-labelled or non-labelled (cold) oligonucleotide probes (Integrated DNA Technologies, Coralville, IA, USA) with sequences shown in Table 1. EMSA experiments were performed as singletons. COS7 cells were untreated, transfected for 24 h with pCMX empty vector or p65-pCMX vector, or treated for 4 h with varying concentrations of recombinant rat TNFα (R&D Systems, Minneapolis, MN, USA). Nuclear protein lysates were obtained with the NE-PER Nucleic Acid and Cytoplasmic Extraction Kit per the manufacturer’s directions (Pierce, Waltham, MA, USA). Lightshift EMSA Optimization and Control Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for control, shift, cold competition, and super-shift reactions, with 1 μL 50% glycerol, 100 mM MgCl₂, and NP-40 included for all EMSA samples. NF-κB p65 antibody (Pierce, Waltham, MA, USA) was used for super-shifts. Samples were run on 6% resolving acrylamide gels and transferred to 0.45 μm Biodyne nylon membranes (Pall Life Sciences, Portsmouth, UK), cross-linked, and visualized (Chemiluminescent Nucleic Acid Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA)).

We followed the method of [32 (link)]. EMSA was analyzed using a chemiluminescent EMSA kit according to the manufacturer's protocol (Thermo Scientific, IL, USA). The biotin upper and lower strands of the Stat3 probe (5′-CTTCATTTCCCGGAAATCCCTA-Biotin-3′ and 5′-TAGGGATTTCCGGGAAATGAAG-Biotin-3′) were synthesized. Nuclear extracts were isolated from two breast cancer cells as described previously [35 (link)]. The biotin-labeled probes were incubated with DHTA-treated nuclear proteins in a final volume of 20 μL EMSA buffer containing poly[dI-dC] for 30 min. The mixtures were electrophoresed on a 6% native PAGE gel in 0.5x TBE at 4°C and visualized using a chemiluminescent nucleic acid detection kit (Thermo Scientific, Waltham, MA USA).