Typhoon fla 9000 image analyzer

Nicotiana tabacum AGO1, VSRs, and the derivatives were expressed in BYL by in vitro translation, mixed (1:1 v/v) and incubated at 25°C for 60 min with 10 nM 21-nt gf698 siRNA duplexes containing 5′ ³²P-labeled guide strand in the additional presence of ATP-regenerating system composed of 0.75 mM ATP, 1 mM MgCl₂, 20 mg/mL creatine phosphate, and 0.4 mg/mL creatine kinase. For AGO2 RISC loading, A. thaliana AGO2 was used with 5′gA siRNA duplex containing guide strand with 5′ adenosine. To analyze RNA, the reaction mixtures were diluted 10-fold with 10 mM Tris, 1 mM EDTA (TE, pH 8.0) and extracted with equal volumes of phenol chloroform isoamyl alcohol (PCI, 25:24:1, v/v). The resulting aqueous phase was recovered, mixed with an equal volume of the native loading dye solution (1× TBE, 10%[v/v] glycerol, bromophenol blue, xylene cyanol), and analyzed in 15% native polyacrylamide gel electrophoresis (PAGE) using 0.5× TBE as running buffer (150 V, 40 min). The signals were detected using BAS-MS imaging plate (FUJIFILM) and Typhoon FLA 9000 image analyzer (GE Healthcare).

The preparation of BYL and in vitro translation reaction were as described previously (Komoda et al. 2004 (link)). Membranous fraction of BYL was pelleted by centrifugation (21,000g, 15 min), and the cytosolic fraction (S20) was used for in vitro translation in this study. All mRNAs were translated at 0.05 µg/µL in reaction mixtures. The expression was measured by the incorporation of ³⁵S-labeled methionine in the translation reaction in the addition of ³⁵S-labeled methionine to the amino acid mixtures excluding methionine (Promega). The reaction mixture was boiled in WB loading buffer (10%[v/v] glycerol, 4%[v/v] SDS, 62.5 mM Tris–HCl [pH 6.8], 5%[v/v] β-mercaptoethanol, and 0.0075% bromophenol blue), and analyzed in NuPAGE 4%–12% Novex Bis-Tris protein gels (BioRad). The signals were detected using BAS-MS imaging plate (FUJIFILM) and Typhoon FLA 9000 image analyzer (GE Healthcare). The relative expression level was calculated by dividing signal intensity of translation product by the number of methionine of each protein.