Figure 4

Xenopus embryonic retinas contain non-CPEB1 cytoplasmic polyadenylation element (CPE)-binding proteins. (A) Western blots using three different antibodies do not detect CPEB1 protein in stage 45 eyes, stage 45 heads, stage 41 eyes, or stage 35/6 eyes. However, they do detect CPEB1 in stage VI oocytes. pc, polyclonal antibody against amino terminus of Xenopus CPEB1; 2B7, monoclonal antibody against human CPEB1; pept, polyclonal antibody against amino-terminal peptide of Xenopus CPEB1 (see Materials and methods). (B) All three antibodies used detect recombinant CPEB1 in stage 24 embryos injected with CPEB1-RBM-GFP mRNA (Figure 3A). MO, morpholino. (C) UV cross-linking with ³²P-labeled CPE-containing probe suggests the presence of non-CPEB1 CPE-binding proteins. Stage 41 eye extracts were incubated with ³²P-UTP labeled probe consisting of the 3' end of Xenopus cyclin B1 with CPE motifs intact (lane 1) or mutated (lane 2), and resolved by SDS-PAGE. Two bands are labeled with ³²P (upper panel; indicated by arrows) but not detected by anti-CPEB1 western blot (lower panel). Proteins binding only to the mutated probe (lane 2) may recognize sequences masked by the CPE-binding proteins in the intact CPE-containing probe. Cross-linked samples were also subject to immunoprecipitation (IP) by control IgG (lane 3) or anti-CPEB1 antibody (lanes 4–5; pc, the polyclonal antibody labeled 'pc' in (A)). Anti-CPEB1 did not immunoprecipitate any cross-linked ³²P-labeled CPE probe (lane 4). The bands at 70 kDa and 50 kDa on the western blot are most likely non-specific, as they do not correspond to any ³²P signal. WB, western blot.