Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 promoted the degradation and polyubiquitination of Snail. (A) UBR5 promoted the proteasomal degradation of Snail. HEK293T cells were transfected with Snail-Flag, Snail 6SA-Flag, UBR5-Myc, GFP, or empty vector and treated with DMSO, chloroquine, MG132, or CT99021 as indicated. The expression of Snail and GFP was assessed by western blotting. (B) UBR5 degraded Snail protein in a concentration-dependent manner. HEK293T cells were transfected with Snail-Flag, GFP, or in combination with different concentrations of wild-type and truncated UBR5-Myc for 48 h. Cell lysates were immunoblotted with anti-Snail antibodies. (C) UBR5 promoted K48 polyubiquitinated chain generation of Snail protein. In cellular ubiquitination assays, UBR5-Myc were co-transfected with Snail-Flag plasmids or with HA-Ub-K63 and HA-Ub-K48 plasmids. Western blotting was performed on cell lysates immunoprecipitated with an anti-Flag antibody, followed by the detection of polyubiquitination levels using an anti-Ub antibody. (D) UBR5 accelerated the Snail protein turnover through the HECT domain. HEK293T cells were transfected with corresponding plasmids. Cells were treated with cycloheximide (CHX) and harvested at indicated time points for immunoblotting with anti-Snail or anti-GFP antibody. The graph shows the quantification of Snail protein levels (based on the band intensity from the gels) normalized to those of GFP over the time course. Snail protein expression at the 0 h time point of treatment with CHX was set as 100 %. Experiments were performed in triplicate, and a representative experiment is presented.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Concentration Assay, Ubiquitin Proteomics, Immunoprecipitation

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 C2768S mutation abrogated the interaction with Snail. (A) His pull-down assays showed the abolished interactions between Snail and the UBR5 C2768S. A schematic representation of the UBR5 wild-type and C2768S mutation. (B) Co-immunoprecipitation assay showed that the interaction between the Snail and the UBR5 C2768S mutation was eliminated. HEK293T cells were transfected with UBR5-Myc, UBR5 C2768S-Myc, and Snail-Flag as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (C) UBR5 C2768S abolished the UBR5-mediated degradation of Snail. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodies. (D) UBR5 C2768S did not accelerate Snail protein turnover. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc and treated with cycloheximide (CHX) as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodi.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot