Figure 4.

RanBP1 mislocalizes to the nucleus in the presence of NES peptide conjugates. (A) The PKI and REV NES peptide conjugates affect the intracellular distribution of RanBP1 but not other tested components of the transport machinery. The indicated peptide conjugates (≈115 ng per nucleus) were injected into oocyte nuclei, and 6.5 hr later the oocytes were dissected. The protein extracts were separated in SDS-containing 10% polyacrylamide gels, and the distributions of specific proteins were analyzed by immunoblotting by using appropriate antibodies and ECL. Oocyte equivalents of the nuclear (N) and cytoplasmic (C) extracts varied for the different proteins: RanBP1, importin β, importin α, and exportin, 3.0 (N) and 0.1 (C); Ran, 1 (N) and 0.5 (C); and RanGAP, 0.5 (N) and 0.1 (C). (B) The PKI NES conjugate, but not the BSA-R conjugate, causes prolonged mislocalization of RanBP1. Oocyte nuclei were each injected with ≈130 ng (MIX and PKI) or ≈190 ng (BSA-R) of peptide conjugates or buffer. Nineteen hours later the oocytes were dissected into nuclear (N) and cytoplasmic (C) fractions. The distribution of RanBP1 in 2 (N) and 0.1 (C) oocyte equivalents was analyzed by immunoblotting as in A.