〔Abstract〕 Objective To study the protein expression and cell localization of RB1-C-terminal phosphorylation site mutants, and to investigate the change of co-localization with Sedlin. Methods The primers of RB1-C-terminal phosphorylation site mutants were designed, and pcDNA3.1-FLAG-RB1-C was used as the template to amplify pcDNA3.1-FLAG-RB1-C-S788 A,S795 A,S807 A,T811 A,T821 A,T826 A. The above mutants were separately transfected into HEK 293 T and COS7 cells, and their protein expression and cell localization were observed by Western blot and immunofluorescence technology; the co-localization of the above mutants and Sedlin were observed by IF technology, and compared with the results of wild type RB1-C. Results The sequencing results showed that the six phosphorylation sites of RB1-C were mutated successfully and expressed normally in eukaryotic cells, which was basically consistent with the cell localization of wild type RB1-C, and they were mainly located in the nucleus. In addition, compared with the wild-type RB1-C, the co-localization of RB1-C-S788 A,S795 A,T821 A,T826 A and Sedlin did not change, that is, they were mainly co-located in the nucleus, but the co-localization of RB1-C-S807 A,T811 A and Sedlin changed significantly, in addition to co-localization and nucleus, there was also significant co-localization in the cytoplasm. Conclusion RB1-C phosphorylation site mutants were successfully constructed and expressed. The mutations of two phosphorylation sites S807 A or T811 A can significantly change the co-localization region of RB1-C and Sedlin in cells, indicating that they are one of the key binding sites of RB1-C and Sedlin, which lays a foundation for further study of the interaction between RB1-C and Sedlin.