Serine pSTAT3 binds to the nucleocytoplasmic transport proteins importin-β1 and CRM1. Total cell, cytoplasmic (Cyt), and nuclear (Nuc) extracts were obtained from CLL cells (CLL 16 and 17), as described in “Isolation of nuclear and cytoplasmic extracts.” Whole-cell extracts, Cyt, and Nuc fractions were immunoprecipitated (I.P.) with anti-STAT3 (A), -importin-β1 (B), and -CRM1 (C) antibodies using protein A–agarose beads. Incubation with beads only (B) was used as a negative control. The immune complex was separated by SDS-PAGE and analyzed by Western immunoblotting using the indicated antibodies. Expression of STAT3, serine pSTAT3, importin-β1, importin-α1, -α3, -α6, -α5 and 7, and CRM1 in total cell extracts of CLL cells was used as a positive control. (D) Freshly isolated CLL 18 cells were incubated for 3 hours with increasing concentrations (2.5-12.5nM) of the CRM1 inhibitor leptomycin B. Nuclear extracts were fractionated and analyzed by Western immunoblotting using anti-STAT3 and antilamin B1 (nuclear protein control) antibodies. Vertical lines indicate realignment of the same gel's image.