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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: Ribosomal L1 domain-containing protein 1 coordinates with HDM2 to negatively regulate p53 in human colorectal Cancer cells

Fig. 6

RSL1D1 Directly Interacts with p53. A GST pull-down assays were performed to evaluate the interaction between full-length RSL1D1 (RSL1D1-FL) and p53 (p53-FL) in vitro. RSL1D1-FL and p53-FL were GST and His tagged, respectively. B, C Schematic diagrams showing p53 (B), RSL1D1 (C), and their truncated variants constructed in this study. D, E GST pull-down assays were performed to map the RSL1D1-binding domain on p53 (D) and the p53-binding domain on RSL1D1 (E). RSL1D1-FL and its truncated variants (RSL1D1-NT and RSL1D1-CT) were GST tagged, whereas p53-FL and its truncated variants were His tagged. F GST pull-down assays were carried out to evaluate the interaction between the RSL1D1-binding domain on p53 and the p53-binding domains on RSL1D1. G Bimolecular fluorescence complementation (BiFC) assay was performed to confirm the interaction between RSL1D1 and p53 in vivo. RSL1D1-FL, RSL1D1-NT, and RSL1D1-CT were cloned into pBiFC-mCherryN159, whereas p53-FL and p53-DBD were cloned into pBIFC-mCherryC160. The recombinant plasmid pairs were co-transfected into HCT116p53+/+ cells. The in vivo interaction between two proteins fused with mCherryN159 and mCherryC160, respectively, was indicated by the red fluorescence in the cells and the nucleus was stained by Hoechst (blue). Co-transfection with empty plasmids pBiFC-mCherryN159 and pBiFC-mCherryC160 was set as a negative control, whereas co-transfection with plasmids pBiFC-mCherryN159-SV40gp6 and pBiFC-mCherryC160-p53 was used as a positive control. Scale bars: 50 μm. H The relative fluorescence intensity in different BiFC groups. To facilitate comparison, the mean value of fluorescence intensity in the cells co-transfected with pBiFC-mCherryN159-SV40gp6 and pBiFC-mCherryC160-p53 was set to 1. Data are represented as mean ± SD. Student’s t test. *P < 0.05 and **P < 0.01 denote significant difference

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