The covalent binding of CBS9106 to Cys528 of CRM1 is reversible. Synthetic peptides (CRM1 WT peptide) containing Cys528 of CRM1 (A) or the mutant peptides (CRM1 MT peptide) in which Cys528 was substituted with Ser (B) were treated with CBS9106 at 37°C for 24 hours and analyzed by MALDI-TOF MS. The black arrowheads correspond to the free peptide, and the white arrowhead corresponds to the adduct of the peptide with CBS9106. (C) RPMI-8226 cells were treated with biotinylated compounds CBS9106-biotin (100nM) or LMB-biotin (0.5nM) for 1 hour, changed to fresh medium, and incubated for the indicated period (0-8 hours). The whole-cell lysates were subjected to pull-down analysis with the use of streptavidin beads. Captured proteins were analyzed by immunoblotting.