FIG. 2.

Two complexes are formed on the stem-loop in Xenopus oocytes. (A) Clones encoding SLBP1 (lane 1) and SLBP2 (lane 2) were transcribed in vitro and then translated in a rabbit reticulocyte lysate in the presence of [³⁵S]methionine. The products were resolved by electrophoresis on an SDS–12% polyacrylamide gel and detected by radioautography. (B) Reticulocyte lysates programmed with either SLBP1 (lanes 2 to 8) or SLBP2 (lanes 9 to 13) mRNAs were incubated with the radiolabeled probe containing the stem-loop, and the complexes were resolved by gel electrophoresis. The antibody to the C-terminal peptide of SLBP1 was added in lanes 5 to 7 and lane 9. The SLBP1 antibody was preincubated with the antigenic peptide in lane 6 and with control peptide from the N terminus of SLBP1 in lane 7. The affinity-purified antibody to SLBP2 was added in lanes 8 and 10. A 100-fold excess of competitor unlabeled RNAs containing the stem-loop (WT) (lanes 3 and 12) or the reverse-stem (RS) (lanes 4 and 13) sequence was added to some reaction mixtures. (C) Oocytes were fractionated into nuclei and cytoplasm, and proteins from three oocytes were resolved by SDS-polyacrylamide gel electrophoresis. The gel was transferred to nitrocellulose and probed with affinity-purified anti-SLBP1 (lanes 1 and 2) or anti-SLBP2 (lanes 2 and 3) antibodies. (D) Total extracts from frog oocytes were incubated with the wild-type stem-loop probe (lanes 1 to 3 and 6 to 8). A 100-fold excess of unlabeled reverse-stem RNA (RS) (lane 2) or wild-type RNA (WT) (lane 3) was included as competitor. The oocytes were fractionated into nuclei (lane 4) and cytoplasm (lane 5). Affinity-purified polyclonal antibodies to SLBP1 (α-X1) (lane 7) or SLBP2 (α-X2) (lane 8) were added to the reaction mixture. The complexes formed with SLBP1 and SLBP2 are denoted x1 and x2, respectively, and the supershifted complexes are denoted SP (lanes 7 and 8). (E) An extract from stage IV oocytes was incubated with the radiolabeled probe and increasing molar amounts of stem-loop competitor (lanes 2 to 6), and the complexes were resolved by gel electrophoresis. A similar extract from stage III oocytes was incubated with increasing amounts of radiolabeled probe (lanes 7 to 9), and the complexes were resolved by gel electrophoresis.