The Y-box binding protein 1 (YB-1) is a multifunctional protein. In the cytoplasm, it is involved in mRNA translation and stabilization, while in the nucleus it participates in replication regulation and DNA transcription. Mostly, YB-1 shows the cytoplasmic localization, but in some cases (under stress or modification) it goes to the nucleus. Its best studied nuclear translocation-causing modification is phosphorylation at S102. The in vitro transport assay technique is a simple and convenient method to study the mechanisms of protein transport. Its advantage is the possibility to control transport conditions. Phosphorylation is a posttranslational modification that regulates many cellular events including protein translocation. To study its effect, a phosphomimetic technique is used, in which amino acid substitutions are introduced into phosphorylation sites to simulate the dephosphorylated state (replacement by Ala) and the phosphorylated state (by Asp or Glu). We used a combination of these two techniques to find out the effect of YB-1 modifications on its nucleus-cytoplasm transport. Methods The site-directed mutagenesis was used to make mutations in the YB-1 phosphorylation sites. The proteins were isolated and the transport assay system was assembled according to [1,2]. For the import reaction, we used YB-1 with S102 replaced by alanine or aspartic acid. Protein localization was assessed by immunofluorescence microscopy. Results In the transport assay system, two types of cell culture conditions were used: standard conditions (YB-1 in the cytoplasm) and transition-stimulating conditions (YB-1 in the nucleus). If phosphorylation stimulates the transition, the phosphomimetic mutant will show the nuclear localization even in standard conditions, while the dephosphorylated one will be detected in the cytoplasm under transition-stimulating conditions. If phosphorylation inhibits the transition, the phosphomimetic mutant will be detected in the cytoplasm in stimulating conditions, while the dephosphorylated one will localize to the nucleus under standard conditions. As expected, YB-1 S102A was absent from the nucleus under transition-stimulating conditions, while YB-1 S102D was detected there in standard conditions. Conclusions Thus, phosphorylation at S102 activates nuclear translocation of YB-1, which is in agreement with the literature data [3,4,5]. Hence, the proposed approach can be used in studies of the posttranslational modification effect on protein transport. ACKNOWLEDGEMENTS. This work was supported by the Russian Foundation for Basic Research (# 18-34-00359\18). REFERENCES 1. Evdokimova V. et al. The EMBO journal. 2001; 20; 5491-5502. 2. Mordovkina D. A. et al. BBRC. 2016; 480; 629-34. 3. Sutherland B. W. et al. Oncogene. 2005; 24; 4281. 4. Basaki Y. et al. Oncogene. 2007; 26; 2736. 5. Kosnopfel C. et al. Molecular Cancer Research. 2018; 16; 1149-60.