CPEB migrates at 65 kDa and 130 kDa in a mild denaturing SDS-PAGE. (A) CPEB immunoreactive 130- and 65-kDa bands were observed when oocyte lysates were prepared with low SDS-containing sample buffer, with sarkosyl instead of SDS, or when the sample was not boiled. (B) Oocyte lysates were prepared with various amounts of SDS, sulfhydryl reducing agent, or RNase to characterize the 130-kDa CPEB immunoreactive band. Left two lanes: The 130-kDa band was sensitive to higher concentrations of SDS, suggesting that it is composed of two or more polypeptides. Middle two lanes: The 130-kDa band was sensitive to the DTT sulfhydryl reducing agent, suggesting that a disulfide bond is involved in protein stabilization. Right two lanes: The 130-kDa band was not sensitive to RNase treatment, suggesting that its formation is RNA-independent. (C) mRNAs encoding HA-CPEB and luciferase were translated in a rabbit reticulocyte lysate supplemented with 35S-methionine. The lysate was then directly applied to SDS-PAGE and visualized by both autoradiography and Western blotting. HA-CPEB migrates as two major species, one at 130 kDa and the other at 65 kDa.
