Sheared salmon sperm dna

Decoy oligonucleotides containing the palindromic p53 cis-element 5′-RRRCWWGYYY-3′ (R = A or G, Y = C or T, W = A or T), which allows self-hybridization and formation of a duplex hairpin that competes with p53 enhancers for binding of transcription factors, were used to inhibit p53-directed transcription in vivo, as previously described by us^{57 (link)}. The sequences of the p53 decoy and control phosphorothioate oligonucleotides (underscored) (Sigma-Aldrich) were as follows: p53 decoy, 5′-GGACATGCCCGGGCATGTCC-3′; control nonsense sequence, 5′-CTAGCTAGCTAGCTAGCTAGCTAG-3′. Cells were seeded at a density of 10⁶ cells onto in 100 mm Petri dishes (Sigma-Aldrich), differentiated as above. After that cells were collected and 800  μl of 2.5 × 10⁷ cell/ml, electroporated in presence of 500 μg sheared salmon sperm DNA (Sigma-Aldrich) and 1 μM of each phosphorothioate oligonucleotide at 330 V and 1000 uF using an Electroporator II (Invitrogen). Next, cells were plated onto 6-well plates (Sigma-Aldrich), grown in a complete medium overnight followed by its replacement with 1% FCIII containing 100 μM OT for 72 h and analysis using RT-PCR as said above.

Nuclear extracts were prepared using the Nuclear Extract Kit (Active Motif) following manufacturer's instructions. Complementary 3′-biotinalayted oligos (sequences in Supplementary Table 1; Life Technologies) were annealed to create substrates for EMSA using the LightShift Chemiluminescent EMSA Kit (Pierce). Reactions contained 170 ng of biotinalayted probe, 3 μg nuclear extract, 3 μg BSA, 10 μg sheared salmon sperm DNA (Sigma), 10 mM Tris, pH 7.5, 1 mM KCl and 2% glycerol. Competitive assays were performed by adding 1–40-fold excess of non-biotinylated probes. Consensus binding sequences for specific transcription factors from Santa Cruz Biotechnology; sc-2505, NFkB; sc-2571, Stat3; sc-2509, Myc-Max; sc-2501, AP-1. The following antibodies (2 μl) were used for super shift experiments: ab31417 and ab31419, c-Jun; ab28837 and ab134067, JunD; ab31421, JunB; sc-7202, c-Fos; sc-150, C/EBP β; sc-372, NFkBp65; sc-66931, IgG; sc-482, Stat3. The abcam antibodies ab31417 and ab134067 were raised against peptides from c-Jun or JunD, respectively, that are less well conserved between the two proteins.

Wells of streptavidin-coated 384-well plates (Pierce, 15506) were washed twice with 100 µL of wash buffer (0.1% Tween-20, 10 mM HEPES, 1.2 mM NaCl, 5 mM MgCl₂, 5 mM KCl, pH 7.4) then incubated with 50 µL of 1 µM biotinylated peptide in peptide binding buffer (1% sheared salmon sperm DNA (Invitrogen, AM9680), 10 mM HEPES, 1.2 mM NaCl, 5 mM MgCl₂, 5 mM KCl, pH 7.4) and were shaken at 500 rpm for 30 min. Wells were then washed six times with 100 µL wash buffer and blocked with blocking solution (100 µL QBlock (Grace Biolabs, 105,106) containing 0.12% Span-80, 0.1% Tween-20, 1% sheared salmon sperm DNA, and 100 µM D-biotin) while shaking at 500 RPM for 30 min. A 25-µL aliquot of fluorescently labeled aptamer probe in binding buffer (wash buffer containing 1% sheared salmon sperm DNA and 300 µM dextran sulfate sodium salt (40 kDa, Sigma-Aldrich, 42867)) was added to each well and allowed to incubate for 60 min while shaking at 500 RPM. Wells were then washed 6–8 times with 100 µL of wash buffer followed by one wash with 100 µL wash buffer without Tween-20 to remove residual bubbles. Finally, 50 µL of wash buffer without Tween-20 was added to each well, and the plate was read using a Tecan Infinite 200 microplate spectrophotometer.