Two rabbit antisera to Aplysia CPEB were raised (BabCO), one against the 17 amino acid C-terminal peptide 644LCNSHQGNYFCRDLLCF660 (CPEB77) and one against the purified recombinant His6-CPEB (463). Purified recombinant protein to Drosophila CG5735R-A was injected into rabbits (BabCO). This rabbit antisera was further affinity-purified against the recombinant protein (Dro 273).
Western Blot membranes were blotted with affinity-purified CPEB 463 (1:4000), affinity-purified anti-CPEB 533 antibody, rabbit affinity-purified Aplysia ubiquitin hydrolase (1:1,500), rabbit anti-syaptophysin (Zymed) (1:3000), mouse anti-human Hsp70 (1:4000) (BD Biosciences), or monoclonal anti-tubulin (1:3000) (Sigma) followed by incubation with 1:10,000 dilution of HRP-coupled anti-rabbit or anti-mouse secondary antibody (Cell Signaling) and visualized by chemiluminescence (Pierce Chemical). For peptide competition, the CPEB77 antibody was preincubated with the peptide (1:20 w/w) at 4°C for 30 min.
For immunohistochemistry fixed cells were stained with 1:30 dilution of affinity-purified CPEB77 (for isolated neurites) or 1:100 dilution of 463 (for one pulse of 5-HT in the synapse) overnight at 4°C.