Requirement for CPEB nuclear localization and RNA binding. (A) CPEB deletion mutants lacking regions between residues 206 and 309 were HA tagged, transfected into NIH 3T3 cells, incubated in the presence of LMB, and analyzed as in Fig. 2. Residues 297–307 were necessary for nuclear localization; each of the 11 residues in this region was changed to alanine and the nuclear localization examined as above. In each case, single alanine substitutions had no effect on nuclear localization. (B) HEK 293T cells were infected with HA-tagged CPEB or CPEB Δ297–307. An extract was then prepared, supplemented with the CPE-containing cyclin B1 3′ UTR, UV irradiated, and subjected to HA immunoprecipitation. The proteins were then analyzed by Western blot for HA (upper panel, two bands are evident; the higher one was likely generated from an upstream cryptic transcription start site of the C-pOZ vector.) and by autoradiography for proteins made radioactive by label transfer (lower panel).
