Pelleted and solubilized ribosomes retain their translational competency. (A) CFE-derived ribosomal pellets P180 generated as described in Figure 2A were resuspended in buffer A by shaking for 30 min at the indicated temperatures. Resuspended RNA was analyzed by northern hybridization with 25S rRNA and 18S rRNA specific probes. (B) The hybridization signals corresponding to the full-length 25S rRNAs and 18S rRNAs were converted to phosphorimaging units and plotted as bar graphs. The error bars represent standard error of the mean (SEM) of three experiments. The differences between the samples were nonsignificant (NS); statistical analysis was performed by one-way ANOVA. (C) 3 µg of resuspended ribosomes were placed into translation reactions containing ribosome-free translational lysate S180 charged with capped TAP-RLuc mRNA (200 ng per reaction). Reaction products were analyzed by the Renilla luciferase assay and the data are presented as bar graphs, wherein error bars represent standard error of the mean (SEM) of three experiments. (D) Proteins and RNA were extracted from the luciferase reactions and further characterized by western blots using anti-TAP antibodies and by northern hybridizations using TAP-specific probe.
