T4 polynucleotide kinase

293T cells were transfected with either 1 μg pcDNA3.1(−) empty vector or C/EBPε expressing vector in 100 mm dishes using X-tremeGENE HP (Roche) according to manufacturer’s instructions. After transfection, cells were cultured for 48 hours before harvesting. Nuclear extracts were prepared from transfected 293T cells, as described48 (link). The following antibodies were used: C/EBPε (sc-158; Santa Cruz); Lamin A/C (#2032; Cell Signaling); and anti-rabbit horseradish peroxidase-conjugated antibody (W4018; Promega). EMSA was performed as described48 (link). Briefly, double-stranded oligonucleotide probes were labeled with [γ-³²P] ATP using T4 polynucleotide kinase (Roche). The following probes were used (C/EBP binding sites are underlined): C/EBP consensus (5′-GATCCATATCCCTGATTGCGCAATAGGC TCAAAA); Trem1 (5′-TGGCCTCACATCCTGTTGTGAAACTTTCCAGAGACTAG); Mutant Trem1 (5′-TGGCCTCACATCCTGCCAAGCCGCTTTCCAGAGACTAG). Labeled probes were incubated with nuclear extracts in 20 mM HEPES, pH 7.5, 200 mM NaCl, 5% Ficoll, 5 mM DTT, 5 mM EDTA, 40 ng/μL of poly-d(I-C), and 40 ng/μL of BSA at room temperature for 20 min. For supershift assays, nuclear extracts were pre-incubated with 200 ng of normal rabbit IgG (sc-2027; Santa Cruz) or anti-C/EBPε (sc-158; Santa Cruz) for 20 min at room temperature prior to the binding reactions. DNA-protein complexes were resolved on native 6% polyacrylamide–TBE gels.

The purified RNA transcripts were treated with rAPid alkaline phosphatase (Roche) in the presence of RNase inhibitor (Promega) at 37°C for 1 h to remove the unlabeled 5′-phosphate. After the inactivation of phosphatase by incubation at 75°C for 2 min, ³²P -γATP (Amersham) and T4 polynucleotide kinase (Roche) were added and the reaction continued for 40 min at 37°C. The ³²P -labeled RNAs were purified by 20% denaturing polyacrylamide gel and recovered by crush and soak procedures. After ethanol precipitation, the labeled RNAs were recovered by centrifugation. To analyze RNA–DNA interactions, labeled RNA probes (10 000 CPM per reaction) were incubated with various amounts of antisense DNA in a final volume of 10 μl of 1× TBE buffer containing 100 mM NaCl and 0.1 mM EDTA. The RNA–DNA complexes were heated at 80°C and annealed for 30 min at 30°C. The reactions were then mixed with 2 μl of 40% sucrose as the loading buffer and loaded into a 20% non-denaturing polyacrylamide gel (19:1 acryl:bisacryl ratio) in 0.5× TBE (Tris-boric acid-EDTA) run at a constant voltage of 150V at 4°C for EMSA analysis. The results were visualized by autoradiography using a Typhoon FLA7000 phosphorimager (GE).

Oligonucleotides utilized for experimentation were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA) and Midland Certified Reagents (Midland, TX, USA). The DNA substrates were 5′ radiolabeled using [γ-³²P]dATP or 3′ radiolabeled using [α-³²P]dCTP (6000 Ci/mmol), purchased from PerkinElmer Life Sciences, Waltham, MA, USA. The Escherichia coli (E. coli) Klenow fragment of DNA polymerase I (for 3′ DNA radiolabeling), the T4 polynucleotide kinase (for 5′ DNA radiolabeling), and ATP were purchased from Roche Applied Science, Indianapolis, IN, USA. All other necessary reagents were purchased from the best available commercial sources.

Radiolabeled probes for EMSAs were generated as described before (43 (link)). Uniformly labeled 126-nt blunt dsRNA was generated by in vitro transcription of T7 promoter-flanked PCR fragments using T7 RNA polymerase in the presence of α-³²P-[UTP]. After annealing of the two radiolabeled RNA strands, unincorporated nucleotides were removed using a G-25 Sephadex column (Roche) and dsRNA was purified from an 8% native polyacrylamide gel. Synthetic 21-nt siRNAs containing 2-nt 3′ overhangs and 19-nt blunt dsRNAs (43 (link)) were end-labeled with γ-³²P-[ATP] using T4 polynucleotide kinase (Roche) and purified on a G-25 Sephadex column.
EMSAs were performed as described previously (11 (link)). Briefly, radiolabeled 126-nt long dsRNA (5 ng), 19-nt dsRNA or siRNA duplexes (1 nM) were incubated with different concentrations of recombinant proteins for 1 h at room temperature. Long dsRNA and siRNA EMSAs were analyzed on 6% and 12% native polyacrylamide gels, respectively. Gels were exposed to a Kodak Biomax XAR film and radioactive signals were quantified with ImageJ software.