gfp tagged proteins  (ATCC)

Journal: Journal of Nuclear Medicine

Article Title: PD-L1 Immuno-PET Reveals Systemic Effects of Localized Oncolytic Virotherapy in a Mouse Model of Head and Neck Cancer

doi: 10.2967/jnumed.125.270922

Figure Lengend Snippet: (A) Representative fluorescent images of MOC1 and MOC2 cells after 48 h of incubation with increasing titers of GFP-expressing RP1-15. GFP shown in gray scale. (B) MOC1 and MOC2 cell viability after 48 h with increasing RP1 titers in vitro, assessed via CellTiter-Glo assay. Significance determined using 1-way ANOVA with Dunnett test. RLU = relative light units.

Article Snippet: The MOC2( PD-L1 ) cell line was generated in our laboratory to overexpress PD-L1 by transducing MOC2 cells with a lentiviral vector carrying the murine CD274 gene tagged with a green fluorescent protein (GFP) reporter (MR 203953L2; OriGene Technologies).

Techniques: Incubation, Expressing, In Vitro, Glo Assay

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Biopanning of a phage library to identify mesothelin-binding peptides. (A) Schematic of biopanning. A phage library was incubated with parental HEK 293T cells; unbound phage was then incubated with HEK 293T cells transfected with a green fluorescent protein (GFP)-tagged mesothelin (MSLN) expression vector (MSLN-HEK 293T). Cell-bound phage was eluted and amplified for the next round. Created with BioRender.com . (B) MSLN-HEK 293T cells 48 h after transfection (green) and parental HEK 293T cells stained with an anti-MSLN antibody (red) and diamidino-2-phenylindole (DAPI) (blue). (C) Phage titer enrichment across screening rounds. pfu, plaque-forming units. (D) Phage-cell-binding ELISA showing binding of individual phage clones to parental versus MSLN-expressing HEK 293T cells. OD, optical density.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Binding Assay, Incubation, Transfection, Expressing, Plasmid Preparation, Amplification, Staining, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Binding of MSLNpep to MSLN-expressing cells. (A) Parental and GFP-tagged (green) MSLN-expressing HEK 293T cells were incubated with tetramethylrhodamine-5-maleimide (TAMRA)-labeled MSLNpep or a control peptide (red) at 4 °C for 1 h and DAPI (blue). (B) Immunoblotting of MSLN expression in AsPC-1 and MIA PaCa-2 pancreatic tumor cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) AsPC-1 and MIA PaCa-2 cells were incubated with fluorescein isothiocyanate (FITC)–MSLNpep (green) at 4 °C for 1 h and an anti-MSLN antibody (red). Nuclei were stained with DAPI (blue), and images were merged. (D and E) AsPC-1 cells were treated with small interfering RNA (siRNA) against MSLN (100 and 200 nM) to knock down MSLN expression or with control (Ctrl) siRNA (200 nM). Cells were incubated with an anti-MSLN antibody (D) or 25 μM FITC–MSLNpep (E) and analyzed by flow cytometry. *** P < 0.001 by one-way ANOVA. (F) AsPC-1 cells were treated with MSLN-siRNA or Ctrl-siRNA (200 nM). After silencing, cells were incubated with 25 μM FITC–MSLNpep (green) at 4 °C for 1 h and an anti-MSLN antibody (red) (1:1,000). Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Binding Assay, Expressing, Incubation, Labeling, Control, Western Blot, Staining, Small Interfering RNA, Knockdown, Flow Cytometry

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Binding of MSLNpep to MSLN protein. (A) HEK 293T and AsPC-1 cell lysates (20, 200, and 400 μg) were incubated with biotin-labeled MSLNpep or control peptide (Ctrlpep) and pulled down with streptavidin-coated magnetic beads. Immunoblotting of the precipitates was performed using an anti-MSLN antibody. (B) Quantification of band intensity (%) for each pull-down relative to input in (A) using ImageJ software. Data are mean ± standard error from 3 independent experiments. *** P < 0.001 by one-way analysis of variance (ANOVA). (C) HEK 293 and AsPC-1 cells were incubated at 4 °C for 1 h with FITC-labeled MSLNpep together with either soluble or mature MSLN. Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (D) HEK 293 and AsPC-1 cells were pretreated with an anti-MSLN antibody at room temperature for 1 h and incubated with FITC–MSLNpep (green) at 4 °C for 1 h. Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (E) ELISA quantifying MSLNpep binding to soluble and mature MSLN. Data are mean ± standard error from 3 independent experiments. ** P < 0.01 by unpaired t test. (F) In silico modeling of MSLNpep binding to MSLN using the PEPFOLD program. MSLN, green; MSLNpep, blue.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Binding Assay, Incubation, Labeling, Control, Magnetic Beads, Western Blot, Software, Staining, Enzyme-linked Immunosorbent Assay, In Silico

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Cytotoxicity of an MSLN-targeted mitochondrial-membrane-damaging peptide. (A) AsPC-1 cells were incubated at 37 °C for 2 h for 1 h with 25 μM FITC-labeled control peptide (Ctrlpep) or MSLNpep (green). Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (B) Diagram of MSLNpep-KLA (L-form) and MSLNpep-kla (D-form). (C to E) AsPC-1 (C), MIA PaCa-2 (D), and HEK 293T (E) cells were incubated with phosphate-buffered saline (PBS), Ctrlpep-KLA, Ctrlpep-kla, MSLNpep-KLA, or MSLNpep-kla for 24 h. After incubation, cell viability was measured using Cell Counting Kit reagents. The half-maximal inhibitory concentration (IC50) values are presented as mean ± standard error from 3 independent experiments. Created with BioRender.com .

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Membrane, Incubation, Labeling, Control, Staining, Saline, Cell Counting, Concentration Assay

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Inhibition of orthotopic pancreatic tumor growth by an MSLN-targeted mitochondrial-membrane-damaging peptide. (A) Treatment schema for mice bearing orthotopic AsPC-1-luc pancreatic tumors. Mice received PBS, Ctrlpep-kla, or MSLNpep-kla at 10 mg/kg body weight. (B) Whole-body bioluminescence imaging on days 0 and 16 after treatment. (C) Total bioluminescent flux after treatment. Data are mean ± standard error ( n = 5). *** P < 0.001; ns, not significant by 2-way ANOVA. (D to F) Total bioluminescent flux for each mouse after treatment with PBS (D), Ctrlpep-kla (E), or MSLNpep-kla (F). (G) Survival rates. (H) Body weights. (I) Serum aspartate aminotransferase (AST) levels. (J) Serum alanine aminotransferase (ALT) levels. (K) Serum alkaline phosphatase (ALP) levels. (L) Serum blood urea nitrogen (BUN) levels. (M) Serum creatinine (CRE) levels. Created with BioRender.com .

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Inhibition, Membrane, Imaging

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Cytotoxicity of an MSLN-targeted mitochondrial-membrane-damaging peptide in human pancreatic cancer patient-derived organoids (PDOs). (A) Immunoblot analysis of MSLN levels in human pancreatic cancer PDOs. Red, MSLN-high organoid; blue, MSLN-low organoid. AsPC-1 and MIA PaCa-2 cells served as controls. (B) Cytotoxicity of MSLNpep-kla in human pancreatic cancer PDOs. Red, MSLN-high organoid; blue, MSLN-low organoid. (C) Correlation between MSLN levels and IC50 values in PDOs ( R 2 = 0.7952). (D) Microscopic morphology of SPT#145 and SPT#144 organoids after treatment with Ctrlpep-kla (100 μM) or MSLNpep-kla (25, 50, and 100 μM). Scale bars, 200 μm.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Membrane, Derivative Assay, Western Blot

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Single-cell expression analysis of MSLN in human pancreatic cancer tissues. Violin plots showing the expression distribution of MSLN across various cell types identified within the pancreatic cancer tumor microenvironment (dataset from Ellerby et al. ). The y -axis represents log-normalized expression levels.

Article Snippet: The pCMV6-AC-GFP mammalian expression vector encoding green fluorescent protein (GFP)-tagged MSLN was purchased from Origene Technologies Inc. (Rockville, MD).

Techniques: Single Cell, Expressing

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