KPNB1, XPO7 and IPO8 mediate the translocation ofNF-κB/p65 into the nucleus.

NF-κB/p65 is retained in the cytoplasm until it is activated in response to stress. Nuclear import of p65 is regulated by importin α in a nuclear localization signal (NLS)-dependent manner. However, the role of importin β family members in the nuclear translocation of p65 is largely unclear. In this study, using high-content siRNA screening, we identified three of 17 importin β family members that are involved in the nuclear import of p65. Our data showed that knockdown of KPNB1, XPO7 and IPO8 reduced the amount of nuclear p65 following tumor necrosis factor-α (TNF-α) stimulation, resulting in lower NF-κB activity. KPNB1 was the major importin β receptor for p65 import, and this import was dependent on the NLS of p65. However, NLS-mutated p65 still entered the nucleus and bound to XPO7 and IPO8. Interestingly, among the six members of the importin α family, KPNA2 was most important for p65 import. Taken together, our results show that the import of p65 mainly relies on the canonical KPNA2/KPNB1 pathway; however, p65 is also imported by an alternative pathway that is independent of its NLS. Redundant importin receptors are likely to maintain the important function of p65 according to need.