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

Journal: bioRxiv

Article Title: Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

doi: 10.1101/2025.09.16.676439

Figure Lengend Snippet: (a) Flow cytometry of fresh whole-blood samples from anonymous donors. C4 protein was quantified (Median Fluorescent Intensity) in major immune cell types using flow cytometry. Jurkat cell line is the negative control and hepG2 is the positive control cell line (top 2 panels in dark gray). Rabbit IgG isotype controls for each major immune cell type are shown in the panel below the C4 protein quantification in that immune cell type (light gray). The bottom panel shows FMO control (dark gray). (b) Median Fluorescence Intensity (MFI) in major immune cell types in ten healthy donors from the Stanford Blood Center, as measured by flow cytometry. Group differences between immune cell types were tested using the nonparametric Mann-Whitney U Test and significance was tested using the false discovery rate (FDR). * = p <0.05, ** = p <0.01, *** = p <0.001. (c) Comparisons that survived threshold for multiple comparisons are shown. A comprehensive list of comparisons is provided in Supplementary Table 3. (f) Average of Median Fluorescence Intensity (MFI) of C4 protein signal in major immune cell types in ten healthy donors from the Stanford Blood Center. An isotype control from the rabbit IgG isotype control for each immune cell type was also provided.

Article Snippet: Then, cells were resuspended in permeabilization buffer and an antibody against the C4 protein beta chain with a conjugated fluorescent tag (Complement C4 polyclonal antibody, AbBy Fluor 647 Conjugated, Bioss, Woburn, MA) for 30 minutes at room temperature with rotation in the dark.

Techniques: Flow Cytometry, Negative Control, Positive Control, Control, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

doi: 10.1101/2025.09.16.676439

Figure Lengend Snippet: (a) Flow cytometry was used to quantify C4 protein in major immune cell subtypes. (b) C4 protein was measured by immunoblotting against C4 protein (22233 Proteintech, Rosemont IL) using WES (capillary-based western blotting) in isolated neutrophils sampled from 15 SCZ and 21 control individuals. Samples were normalized using cellular actin (8H10D10, Invitrogen, Waltham, MA, USA). (c) Table showing the Descriptive statistics of measured immune cell-associated C4 protein in samples from individuals with SCZ compared to controls using different methods. Group comparisons of C4 protein for each major immune cell type (and plasma) were performed using one-way ANOVA. (d-f) CM and NCM were isolated from frozen live PBMC samples. Isolated cells were incubated, fixed, and stained for C4 protein (antibody directed against C4 protein, Proteintech, 22233-1-AP). C4 protein is localized throughout monocyte cells but is preferentially localized at the periphery. Representative images of CMs stained with Hoeschet stain for DNA and fluorescently labeled antibody against C4 protein in a sample from a control participant and a sample from a participant with SCZ. (e) Quantification of C4 protein throughout each cell was performed by measuring the Mean Intensity (Mean) from immunofluorescent images of C4 protein using Fiji. A mask was created from the nuclear stain and used to subtract the central C4 protein fluorescence to determine the Peripheral C4 protein Mean Fluorescence. (f) Table showing the exploratory descriptive statistics of CM and NCM C4 protein in samples from individuals with SCZ compared to controls. RFU = Relative Fluorescent Unit.

Article Snippet: Then, cells were resuspended in permeabilization buffer and an antibody against the C4 protein beta chain with a conjugated fluorescent tag (Complement C4 polyclonal antibody, AbBy Fluor 647 Conjugated, Bioss, Woburn, MA) for 30 minutes at room temperature with rotation in the dark.

Techniques: Flow Cytometry, Western Blot, Isolation, Control, Clinical Proteomics, Incubation, Staining, Labeling, Fluorescence