Fig. 1

Cloning of ApCPEB4 and its expression in the CNS. a Amino acid sequence of a cloned full-length ApCPEB4. Sequence analysis showed that ApCPEB4 had two conserved RRMs (underlined), one conserved PKA phosphorylation sites (box). b Alignment of RRM domain of Aplysia CPEB4 (ApCPEB4), mouse CPEB3 (mCPEB3), mouse CPEB1 (mCPEB1) and Aplysia CPEB (ApCPEB). c The phylogenetic relationship between CPEBs in different species was determined by ClustalW. d mRNA structure of the ApCPEB4. ApCPEB4 contains ~20 bp 5′UTR (untranslated region), ~2 kb open reading frame (ORF), and ~1 kb 3′UTR. Arrowed inset indicates the detailed nucleotide sequence of the 3′UTR. Blue underline indicates hexanucleotide sequence (AATAAA). e Expression pattern of ApCPEB4 mRNA. RT-PCR of total RNA (1 μg) isolated from Aplysia CNS, ovotestis, or gill with gene-specific primers. Aplysia housekeeping gene S4 was used as a control for the amplification. f Western blotting of ApCPEB4 using purified GST-fused ApCPEB4 or total lysates from various tissues including pleural ganglion, buccal ganglion and ovotestis. g A representative Western blot (left) and quantification (right) of ApCPEB4 in Aplysia pleural ganglia extracts prepared from pleural-to-pedal ganglia exposed to 5 times of 5 min treatment of 5-HT. Total extracts were prepared at indicated times and 20 μg of proteins were blotted with anti-ApCPEB4 antibodies (left, top panel). The same extracts were also blotted with anti-tubulin antibodies as loading controls (left, bottom panel). 5-HT treatment significantly increased the level of ApCPEB4 in the extracts. **, p < 0.01, two-tailed unpaired t test. h One microgram of total RNA from pleural ganglia was used for RT-PCR with gene-specific primers. As a stimulation control, we used ApC/EBP, an immediate early gene. ApC/EBP was transcriptionally enhanced in response to 5-HT stimuli. Aplysia S4 was used as an amplification and loading control. *, p < 0.05 compared to that of control ApC/EBP, two-tailed unpaired t test