Journal: ACS Applied Bio Materials

Article Title: Zinc-Mediated Loading and Release of His-Tagged Recombinant Proteins in Self-Assembling Peptide Coacervates

doi: 10.1021/acsabm.5c02044

Figure Lengend Snippet: Loading capacity and specificity of His-tagged green fluorescent protein (GFP) in Zn 2+ :EAKH6 coacervates and controls. (A) Pulldown assay based on the centrifugation of GFP and metal fibrils. Fluorescence measured in fibril composites of Zn 2+ or Ca 2+ at metal-to-peptide molar ratios of (B) 10:1 and (C) 1:1, and controls, including (D) EAK, (E) with EDTA, and (F) with excess imidazole (240 mM). Unpaired t -test: **** p < 0.0001, *** p < 0.0001, ** p < 0.001, and * p < 0.01. Schematic (A) was created in BioRender via an academic license.

Article Snippet: Green Fluorescent Protein coexpressed with a hexahistidine tag (His-tag) (HisGFP) was purchased from Sino Biological.

Techniques: Centrifugation, Fluorescence

Journal: ACS Applied Bio Materials

Article Title: Zinc-Mediated Loading and Release of His-Tagged Recombinant Proteins in Self-Assembling Peptide Coacervates

doi: 10.1021/acsabm.5c02044

Figure Lengend Snippet: In vivo retention of pG-His injected subcutaneously into the mouse footpad. (A) Fluorescent images of the footpad from 0 to 120 h obtained at 700 nm, with a resolution of 170 μm. (B, C) Quantification of fluorescent intensities in mice injected with dye-conjugated pG-His formulated in (B) saline or (C) Zn 2+ :EAKH6. (D) Retention of pG-His ( n = 3). Unpaired t -test with * p < 0.01.

Article Snippet: Green Fluorescent Protein coexpressed with a hexahistidine tag (His-tag) (HisGFP) was purchased from Sino Biological.

Techniques: In Vivo, Injection, Saline

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: Journal of Molecular and Cellular Cardiology Plus

Article Title: Rab10 plays a protective role in the development of pathological cardiac hypertrophy

doi: 10.1016/j.jmccpl.2025.100494

Figure Lengend Snippet: Rab10 overexpression inhibited Ang II-induced cardiomyocyte hypertrophy and Ang II-initiated signaling pathway. (A) Western blot analysis of exogenous Rab10 in NRCMs infected with Ad-GFP or Ad-Rab10 with anti-Myc or anti-Rab10 antibody. Quantification of the relative Rab10 level (lower, n = 3, three independent replicates). (B) Representative images of NRCMs infected with Ad-GFP or Ad-Rab10 and then treated with 200 nmol/L Ang II for 24 h. Hypertrophy was assessed by cell surface area measurements ( n = 3, 150 cells counted per experiment; right). (C) qPCR analysis of the ANP mRNA level in NRCMs ( n = 3, three independent replicates). (D) Representative immunoblotting analysis of p-ERK1/2, ERK1/2, p-AKT and AKT in NRCMs infected with Ad-GFP or Ad-Rab10 and treated with 200 nmol/L Ang II ( n = 3, three independent replicates). (E) Quantification of the relative protein levels (right). β-actin was used as a loading control. Data are presented as mean ± s.e.m., and n represents number of samples. * P < 0.05 , ** P < 0.01 versus Ad-GFP or saline; # P < 0.05, ## P < 0.01 versus Ad-GFP plus Ang II.

Article Snippet: Recombinant adenoviruses expressing the green fluorescent protein (GFP) alone (Ad-GFP), the full-length Rab10 cDNA with a C-terminal Myc-tag (Ad-Rab10), driven by the cytomegalovirus promoter were generated using AdEasy (MP Biomedicals).

Techniques: Over Expression, Western Blot, Infection, Control, Saline