Electrophoretic mobility shift assays

RNA was 5′-end labeled using T4 polynucleotide kinase and [γ-³²P]ATP and then it was purified by denaturing polyacrylamide gel electrophoresis (PAGE). The RNA concentration was determined by the UV absorbance at 260 nm. RNA was annealed by heating at 90 °C and slow cooling to room temperature in sterile water. RNA, helicates, and the ADP-1 peptide were mixed in a buffer (5 μL) containing Tris-HCl (50 mM, pH 8.0), KCl (50 mM), DTT (100 mM), and Triton X-100 (0.05%) and incubated for 10 min on ice. The samples were analysed by electrophoresis on 15% polyacrylamide (PAA) gels in 0.5 × TB buffer and electrophoresed at 5 W and 5 °C. Gels were exposed to a phosphor imaging plate and scanned with a FUJIFILM BAS-2500 bio-imaging analyzer.