Nylon membrane

Nuclear protein extraction was carried out as previously described [21 (link)]. A double-stranded NFκB DNA probe (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) was end labeled with biotin. Detection of NFκB-oligonucleotide complex was performed using a LightShift chemiluminescent EMSA kit (Pierce, Northumberland, UK). Briefly, nuclear protein (5 μg) was incubated with 20 fmol of biotin-labeled oligonucleotide for 20 min at room temperature in binding buffer consisting of 10 mM Tris at pH 7.5, 50 mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl₂, 50 ng of poly(dIdC), and 0.05% Nonidet P-40. The specificity of the NFκB DNA binding was determined in competition reactions in which a 200-fold molar excess of unlabeled wild type or mutant NFkB probe (5′-AGT TGA TAT TAC TTT TAT AGG C-3′) was added to the binding reaction. Products of binding reactions were resolved by electrophoresis on a 6% polyacrylamide gel. NFκB-oligonucleotide complex was electroblotted onto a nylon membrane (Amersham). After incubation in blocking buffer for 1 hour at room temperature, the membrane was incubated with streptavidin-HRP conjugate for 30 min at room temperature. The membrane was incubated with chemiluminescent substrate for 5 min and exposed to radiographic film.

Genomic DNA was extracted with a TIANamp Genomic DNA Kit (DP304-03, Tiangen), except that samples were treated without RNase A according to the manufacturer's instructions. After quantification by a UV/VIS spectrophotometer (UV5Nano, Mettler Toledo), 1 mg of DNA from each sample was blotted on a nylon membrane (Amersham) with a dot blot apparatus and vacuum suction. For RNase H treatment, 1 mg of DNA was incubated with RNase H at 37°C overnight, then extracted with a TIANamp Genomic DNA Kit. The membrane was denatured for 10 min in a solution containing 0.5 M NaOH and 1.5 M NaCl, and neutralized in a solution containing 1 M NaCl and 0.5 M Tris–HCl pH 7.0 for another 10 min. Before being completely dried, the membranes were cross-linked with UV (1200 mJ/cm²) and stained in 1% methylene blue solution (G1303, Solarbio) for 10 min. Subsequently, the membranes were washed with TBST buffer, blocked in 5% (w/v) milk in TBST buffer for 1 h at room temperature and incubated with S9.6 antibody (1:500) overnight at 4°C. After several washes in TBST buffer, the blots were incubated with IgG secondary antibody (1:1000) in TBST buffer for 1 h at room temperature. The blots were visualized using chemiluminescence on a Tanon Imaging System and quantified by ImageJ software.

TRF analysis was performed as previously described^{65 (link)}. In brief, genomic DNA was isolated by phenol–chloroform extraction and digested with AluI and MboI overnight. A total of 4 µg of digested gDNA was separated on 0.7% agarose gel at 40 V and transferred to a positively charged Nylon membrane (Amersham, RPN203B). After cross-linking the DNA and prehybridization (5× SSC. 0.1% N-lauroylsarcosine sodium salt solution, 0.04% SDS) for 2 h at 65 °C, the membrane was incubated with digoxigenin-labelled TelG probe diluted in hybridization buffer (1.3 nM final concentration) overnight at 65 °C. Digoxigenin-labelled TelG probe was generated as previously described^{66 (link)}. Then, the membrane was washed three times with wash buffer 1 (2× SSC, 0.1% SDS), one time with wash buffer 2 (2× SSC) for 15 min each and blocked in freshly prepared blocking solution (100 mM maleic acid, 150 mM NaCl, pH 7.5, 1% (w/v) blocking reagent (Roche, 11096176001)) for 30 min. Next, the membrane was incubated for 30 min in anti-digoxigenin-AP antibodies (Roche, 11093274910) diluted in blocking solution, washed twice in wash buffer 3 (100 mM maleic acid, 150 mM NaCl, pH 7.5, 0.3% (v/v) Tween-20) for 15 min each and equilibrated in AP buffer (100 mM Tris, 100 mM NaCl, pH 9.5) for 2 min. Digoxigenin-labelled telomeric DNA was detected using CDP-star ready to use (Roche, 12041677001) solution.

Total small RNAs extracted from indicated tissues using a mirVana miRNA isolation kit (Ambion, USA) were separated by electrophoresis on a 15% polyacrylamide gel containing 7 M urea for 2 h at 5 mA, then transferred to a nylon membrane (Amersham Biosciences, UK) at 300 mA for 2 h. Subsequently, the membrane was cross-linked by UV and then prehybridized in a prehybridization solution (Roche, Switzerland) for 30 min. To detect the target miRNA, the membrane was hybridized with digoxigenin (DIG)-labeled shrimp miR-965 probe (5′-GAGGGGAAAAGCCATACGCTTA-3′) overnight. After that, the membrane was rinsed and then blocked in a blocking solution (Roche, Switzerland) for 1 h at room temperature. Lastly, the membrane was incubated with the antibody against DIG-labeled alkaline phosphate (Roche, Switzerland) for 2 h, and the target miRNA was visualized with the substrate BCIP/NBT solution (Roche, Switzerland).

The 197 bp probe containing -196 to +1 PsaA promoter region was PCR amplified and labeled at the 3′-end with biotin-14-dCTP using biotin labeling kit (Invitrogen) according to the manufacturer’s instructions. DNA–protein interactions were performed in 25 mcL reactions containing following reagents: 2.5 mcL of × 10 binding buffer (100 mM Tris HCl, 250 mM KCl, and 10 mM DTT), 1 mcg poly dIdC (Sigma–Aldrich), 2,5% glycerol, 0.05% Triton X-100, 5 mM MgCl2, 10 mM EDTA. The reaction mixture was incubated with DNA and protein at room temperature for 30min and was run on 6% native TBE-PAGE in x0,5 TBE buffer at 100 V. DNA was transferred to nylon + membrane (Amersham) and was UV cross-linked to the membrane, incubated with Streptavidin-HRP and detected by Chemoluminescence Nucleic Acid Detection Module (Pierce) according to manufacturer’s instructions (Shaikhali et al., 2012a (link),b (link)).

The genomic DNA of 30 μg was digested completely with Hinde, separated by 0.8% gel electrophoresis, transferred into a nylon membrane (Amersham, United Kingdom), and hybridized with digoxin-labeled DNA fragments of g10evo at 65^°C overnight. The signaling was detected by the image analyzer FLA-5100 (FUJIFILM, Japan). Detailed procedures were as described in the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Switzerland).