Functions in nuclear protein import, either in association with an adapter protein, like an importin-alpha subunit, which binds to nuclear localization signals (NLS) in cargo substrates, or by acting as autonomous nuclear transport receptor. Acting autonomously, serves itself as NLS receptor. Docking of the importin/substrate complex to the nuclear pore complex (NPC) is mediated by KPNB1 through binding to nucleoporin FxFG repeats and the complex is subsequently translocated through the pore by an energy requiring, Ran-dependent mechanism. At the nucleoplasmic side of the NPC, Ran binds to importin-beta and the three components separate and importin-alpha and -beta are re-exported from the nucleus to the cytoplasm where GTP hydrolysis releases Ran from importin. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus. Mediates autonomously the nuclear import of ribosomal proteins RPL23A, RPS7 and RPL5. In association with IPO7, mediates the nuclear import of H1 histone. In vitro, mediates nuclear import of H2A, H2B, H3 and H4 histones. In case of HIV-1 infection, binds and mediates the nuclear import of HIV-1 Rev. Imports SNAI1 and PRKCI into the nucleus.
Background References
1. Dean K.A., von Ahsen O., Goerlich D., Fried H.M. Signal recognition particle protein 19 is imported into the nucleus by importin 8 (RanBP8) and transportin. J. Cell Sci. 114:3479-3485 (2001)
2. Choi S., Yamashita E., Yasuhara N., Song J., Son S.Y., Won Y.H., Hong H.R., Shin Y.S., Sekimoto T., Lee S.J. Structural basis for the selective nuclear import of the C2H2 zinc-finger protein Snail by importin beta. Acta Crystallogr. D 70:1050-1060 (2014)
Western blot analysis of KPNB1 on different lysates with Rabbit anti-KPNB1 antibody (HA721702) at 1/1,000 dilution.
Lane 1: HeLa cell lysate (20 µg/Lane) Lane 2: HepG2 cell lysate (20 µg/Lane) Lane 3: A431 cell lysate (20 µg/Lane) Lane 4: A549 cell lysate (20 µg/Lane) Lane 5: Jurkat cell lysate (20 µg/Lane) Lane 6: K-562 cell lysate (20 µg/Lane) Lane 7: Caco-2 cell lysate (20 µg/Lane) Lane 8: NIH/3T3 cell lysate (20 µg/Lane) Lane 9: PC-12 cell lysate (20 µg/Lane) Lane 10: C6 cell lysate (20 µg/Lane) Lane 11: Mouse spleen tissue lysate (40 µg/Lane) Lane 12: Mouse testis tissue lysate (40 µg/Lane) Lane 13: Rat spleen tissue lysate (40 µg/Lane) Lane 14: Rat testis tissue lysate (40 µg/Lane)
Predicted band size: 97 kDa Observed band size: 97 kDa
Exposure time: 1 minute;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721702) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature.
KPNB1 was immunoprecipitated in 0.2mg HeLa cell lysate with HA721702 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA721702 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: HeLa cell lysate (input) Lane 2: Rabbit IgG instead of HA721702 in HeLa cell lysate Lane 3: HA721702 IP in HeLa cell lysate