Poly di dc

EMSA was performed to detect NF-κB activation in hippocampal homogenates obtained from the brains of vehicle-, Aβ-, and pentamidine-treated mice. Double stranded oligonucleotides containing NF-κB recognition sequence (5′AGTTGAGGGGACTTTCCCAGGC-3′) were end-labeled with ³²P-γ-ATP (Amersham, Milan, Italy). Nuclear extracts were incubated for 15 min with radiolabeled oligonucleotides (2.5–5.0 × 10⁴ cpm) in 20 mL reaction buffer containing 2 mg poly dI-dC (Boehringer-Mannheim, Milan, Italy), 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM dl-dithiothreitol, 1 mg/mL bovine serum albumin, and 10% glycerol. Nuclear protein-oligonucleotide complexes were resolved by electrophoresis on a 6% nondenaturing polyacrylamide gel in 1X Tris Borate EDTA buffer at 150 V for 2 hrs at 4°C. The gel was dried and autoradiographed with an intensifying screen at −80°C for 20 h. Subsequently, the relative bands were quantified by densitometric scanning with Versadoc (Bio-Rad Laboratories) and computer software (Quantity One Software, Bio-Rad Laboratories). Oligonucleotide synthesis was performed to our specifications by Tib Molbiol (Boehringer-Mannheim, Ingelheim am Rhein, Germany).

25μg of nuclear extract was incubated with 3μg BSA (New England Biolabs) and 1ug poly(dIdC, Roche) in 2x buffer (12% glycerol, 24mM HEPES, 8mM TrisHCl pH 8.0, 2mM EDTA, 1mM DTT) on ice for 10 minutes. In competition assays, non-labeled oligos were added to the reaction before the 10 minute incubation. Then 4,000 counts per million (cpm) of probe was added and incubated on ice for 20 minutes.

EMSA reactions were set up on ice to a final volume of 6.5 μL. Into each reaction was added the appropriate volume of purified VapBC protein, 0.4 ng DIG-labelled DNA, 2 μl 5x Binding Buffer (Roche), 0.5 μl 1 mg.mL^-1 poly dI-dC (Roche) and ultrapure water sufficient to adjust the total volume to 6.5 μl. Binding reactions were incubated for 20 minutes at room temperature. Reactions were placed back on ice and 2.5 μl loading buffer with bromophenol blue (Roche) was added. Tris-borate-EDTA gels (6%) were pre-run in 1 x TBE buffer at 60 V for 20 minutes. Samples were loading and run at 150 V until the dye-front had migrated approximately two-thirds of the way down the gel. Contact blotting was performed for 30 minutes using Whatman 2 MM blotting paper, after which the DNA was crosslinked to a Hybond-N⁺ (GE Healthcare) membrane using a Bio-Link BLX-254 BRL UV Crosslinker Irradiation System (Life Technologies) which irradiated the membrane with 120 mJ at UV 254 nm for 20 seconds. DNA was detected by a chemiluminescent immunoassay and visualised on films.

BacR qPCR reactions were performed in a total reaction volume of 15 µL containing 1x Kapa Probe Fast (PeqLab, Erlangen, Germany), 100 nM BacR_f, 500 nM BacR_r, 100 nM BacR_p, and 2.5 µL of DNA template. The amplification reactions were run on a 7500 Fast Real-Time PCR system (Applied Biosystems, New York, NY) according to the following thermal cycling conditions: an initial step of 5 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and 15 s at 60 °C. Unless otherwise noted, all amplification reactions were performed in triplicate, including no-template controls (NTC) in each run to check for contamination. The results of qPCR were scored negative when the c_q value was undetermined or <1 copy was detected. Quantification was based on dilutions of a plasmid standard carrying a single copy of the BacR marker sequence. Plasmid standard dilutions were prepared in an unspecific background of 500 µg L⁻¹ poly(dI-dC) (Roche Diagnostics, Mannheim, Germany) to avoid adsorption of plasmid DNA onto the reaction tubes at low concentrations. A total of seven tenfold serial dilutions of plasmid standard (10⁰–10⁶ marker copies per reaction) was included in each qPCR run. In addition, DNA sample dilutions were measured in each qPCR run and were judged free of inhibition based on matching concentrations between two dilutions, as described previously⁴⁴ .

Nuclear extracts were prepared as described previously and stored at −80°C until use [13 (link), 29 (link)]. EMSAs were performed essentially as described previously [13 (link)]. DNA probes for EMSAs were synthesized as oligonucleotides. The sequences of the individual oligonucleotides in the sense orientation were as follows: probe 1, 5′-AGTGTGTGCCGGGAAAAGGCTCATA-3′ (nt. −148~−124 of human REG Iα promoter; nt. 1048–1072 of J05412), probe M1, 5′-AGTGTGCAGTAGGAAAAGGCTCATA-3′.
DNA-protein binding reactions were performed by incubation of the nuclear extracts in a solution containing 10 mM HEPES (pH 7.8), 50 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 10% glycerol, 5 mM DTT, 1 mg/mL BSA, 0.7 mM PMSF, 50 ng/μL of poly(dI-dC) (Roche) for 10 min at room temperature, followed by an additional 30 min incubation with ³²P-end-labeled probe at room temperature. For supershift assays, 1 μg of anti-signal transducer and activator of transcription (STAT)3 antibody (Santa Cruz Biotechnology, SC-482X, Santa Cruz, CA) [30 (link)] was added to the samples and incubated for 15 min before incubation with the labeled probe. DNA-protein complexes were separated on 4% nondenaturing acrylamide gels and detected by autoradiography.