Texas red maleimide

The preparation of cell extracts was performed based upon a previous study (37 ). Briefly, cultured cells were washed twice with ice-cold PBS and scraped with a plastic cell scraper in 1ml of ice-cold PBS. Cells were spun in a microfuge for 5 min. at 700 × g, at 4 °C. Then, 100 μl of ice-cold lysis buffer containing 1% Triton ×100, 25 mM Tris-HCl (pH7.4) and 40 mM KCL per 10⁷ cells was added and incubated for 20 min. on ice. Samples were spun at full speed in a microfuge at 4°C and supernatant was collected. Pre-let-7 (PM12304, PM10050, Thermo Fisher Scientific) was labeled with Texas-red fluorescence using the 5′ EndTag Labeling DNA/RNA kit and Texas Red Maleimide (Vector Laboratory), according to the manufacturer’s instructions. Binding reactions were conducted with 1 ng of Texas-Red labeled pre-let-7 in a total volume of 20 μl together with 50 μg of yeast tRNA and indicated amounts of protein extracts. The labeled RNA was incubated at 37°C for 2 min and cooled on ice for 3 min. The binding buffer contained 20mM Tris/HCL, pH7.5, 60mM KCL, 20U of RNase inhibitor (Thermo Fisher Scientific, #10777019) and 1mM DTT. After 30 min of incubation at room temperature, 4 μl of loading buffer was added to the EMSA sample and resolved on a DNA retardation gel (EC6365BOX, Thermo Fisher Scientific). RNA bands were visualized using ChemiDoc™ MP Imaging System (Bio-Rad).