Image analyzer

Conditioned media was collected after 24 h stimulation, mixed with non-reducing sample buffer, and electrophoresed on a polyacrylamide gel containing 0.1% (w/v) gelatin. The gel was washed at room temperature for 30 min with 2.5% (v/v) Triton X-100 solution, and subsequently incubated at 37°C for 16 h in 5 mM CaCl₂, 0.02% (v/v) Brij, and 50 mM Tris–HCl (pH 7.5). The gel was stained for 30 min with 0.25% (w/v) Coomassie Brilliant Blue in 40% (v/v) methanol/7% (v/v) acetic acid and photographed on an image analyzer (Fuji-Film). Proteolysis was visualized as a white zone in a dark blue field. Densitometric analysis was performed using Multi Gauge Image Analysis software (Fuji-Film).

Recombinant LGP2 proteins were mixed with ³²P-labeled synthetic dsRNA (25/25c) [49] (link) in a reaction mixture (20 mM Tris-HCl (pH 8.0), 1.5 mM MgCl₂, and 1.5 mM DTT) in the presence or absence of recombinant Pumilio proteins. After incubation at 37°C for 15 min, the reaction mixture was applied to a 15% acrylamide gel (TBE buffer) and the radioactivity was detected with an Image Analyzer (FUJIFILM, Tokyo, Japan).

OP9 cells were pretreated with 40 μg/mL AOE for 1 h and then differentiated at 37°C. Cells were lysed with ice-cold mammalian protein extraction reagent (M-PER) (Pierce Biotechnology, Rockford, IL, USA), and the protein concentration in the lysate was determined using the Bradford method. Samples (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 10% acrylamide and transferred to Hybond-P polyvinylidene fluoride membranes (GE Healthcare Life Sciences, Buckinghamshire, UK) using a western blot apparatus. Each membrane was blocked for 2 h with 2% bovine serum albumin or 5% skim milk and then incubated overnight at 4°C with 1 μg/mL of a 1 : 2,000 dilution of primary antibody. HRP-conjugated IgG (1 : 2,000 dilution) was used as the secondary antibody. Protein expression levels were determined by signal analysis using an image analyzer (Fuji-Film, Tokyo, Japan).