Figure 4. The synthesis of select ribosomal subunits is decreased in an age‐dependent manner in K3 mice.
- Western blot analysis of de novo synthesised proteins demonstrates age‐dependent alterations in protein synthesis in K3 mice compared with WT controls. AHA‐labelled proteins from 2‐ and 5‐month‐old K3 and WT mice were purified using BONCAT and probed by Western blotting (BONCAT‐WB). The synthesis of ribosomal proteins RPLP0, RPL19 and RPS16, as well as histone H3, was found to be significantly decreased in K3 mice, whereas that of histone H4 was increased and that of synapsin I was unaltered (two‐way ANOVA, Sidak's multiple comparison test, n = 3 animals).
- Schematic representation of the FUNCAT‐PLA (proximity ligation assay) technique, showing that newly synthesised, AHA‐labelled RPL23 is biotinylated using DIBO‐biotin and subsequently co‐labelled with anti‐biotin and anti‐RPL23 primary antibodies. Co‐labelling is then detected using PLA antibodies, which results in a fluorescent signal when the two targets are in close proximity (i.e. < 40 nm apart).
- Synthesis of RPL23 in K3 mice is decreased in an age‐dependent manner. Compared to WT mice, significantly lower numbers of RPL23 FUNCAT‐PLA punctae are detected in layer 2/3 cortical neurons of K3 mice at both 2 and 5 months of age (two‐way ANOVA, Sidak's multiple comparison test, n = 3 animals).