5.3.6. rRNA N-Glycosylase Assays on Rabbit Reticulocyte, Yeast, Bacterium Lysates, and HeLa Cells

rRNA N-glycosylase assays were conducted as described elsewhere [16]. Rabbit reticulocyte lysate (40 μL) was incubated with 5 μg of RIP at 30 °C for 1 h. N-glycosylase activity on Saccharomyces cerevisiae ribosomes was assayed in 50 μL samples of S−30 lysate from yeast in 10 mM Tris–HCl buffer (pH 7.6) containing 10 mM KCl, 10 mM magnesium acetate, and 6 mM 2-mercaptoethanol, which was incubated with 5 μg of RIPs at 30 °C for 1 h. N-glycosylase activity on Micrococcus lysodeikticus ribosomes was assayed using 100 μL of bacterial lysate samples in 20 mM Tris–HCl buffer (pH 7.8), which were incubated with 5 μg of RIP at 30 °C for 1 h. After treatment, the RNA was extracted by phenolization, treated with 1 M aniline acetate (pH 4.5), and precipitated with ethanol. HeLa cells (1 × 10⁶/plate) were incubated for 48 h in the presence of 40 nM of nigrin l. After treatment, cells were harvested by centrifugation at 1000× g for 5 min. The pellets were lysed, and the RNA was isolated following the instruction of the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). RNA was treated with 1 M aniline acetate (pH 4.5) for 10 min at 0 °C and precipitated with ethanol. The RNAs were subjected to electrophoresis at 15 mA for 2 h (rabbit and HeLa cells) or 1 h 30 min (yeast and bacterium) in a 7 M urea/5% (w/v) polyacrylamide gel and stained with GelRed nucleic acid stain (Biotium Inc., Hayward, CA, USA) [16].