Fig 4.

Deactivation of CRM1 by 4-OI-mediated C528 alkylation. (A) Mass spectrometry screening of 2,3-dicarboxypropylated cysteine residues in THP1 cells treated with 4-OI. Cells were treated with 250 µM 4-OI, and IA-DTB-based competitive cysteine profiling was performed to identify 2,3-dicarboxypropylated cysteine residues on target proteins. Highlighted are C288 on Keap1, as well as C528, C119, and C164 on CRM1. Mass spectrometry data are representative of three independent experiments. (B) CRM1 protein expression levels in Jurkat and Jurkat XPO1^C528S cells. The cells were treated with either DMSO, 250 µM 4-OI, or 25 nM KPT-330 for 24 hours. CRM1 protein expression levels were assessed by Western blotting, and β-actin was used as loading control. Blots are representative of four individual experiments. (C) Expression levels of p65 and p53 protein in nucleus, cytoplasm, and whole-cell fractions of Jurkat and Jurkat XPO1^C528S cells upon treatment. The cells were treated with DMSO, 250 µM 4-OI, or 100 nM KPT-330 for 2 hours, and nuclear-cytoplasmic fractionation was performed. H3 was used as loading control for nuclear fractions and β-actin for cytoplasmic and whole-cell fractions. Blots are representative of four (p65) or three individual experiments (p53). (D) Densitometry of blots shown in panel B. Protein levels have been normalized relative to the untreated control of the respective cell line. (E and F) Densitometry of blots shown in nuclear fraction of panel C. Protein levels have been normalized relative to the untreated control of the respective cell line.