Journal: ACS Synthetic Biology

Article Title: High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB

doi: 10.1021/acssynbio.0c00130

Figure Lengend Snippet: (a) MALDI-TOF MS data of CS5 before (blue) and after (red) treatment with CDAP and hypothetical structure of CS5. (b) SDS-Tricine gel of CS5 coexpressed with NisBTC or NisT before (&) and after (#) streptavidin column purification. (c) LC-MS/MS data of CS5(13–20). The observed fragments demonstrate that the cyclic strep ligand was formed.

Article Snippet: Aliquots of OD 600 = 3.0 from these cell suspensions were incubated with a 4000-fold diluted horseradish peroxidase (HRP)-conjugated streptavidin solution (Thermo Fisher Scientific, Waltham, MA) in a final volume of 1 mL of MES-Ca plus 0.2% Tween 20 and 1% BSA at room temperature for 1 h under rotation.

Techniques: Purification, Liquid Chromatography with Mass Spectroscopy

Journal: ACS Synthetic Biology

Article Title: High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB

doi: 10.1021/acssynbio.0c00130

Figure Lengend Snippet: (a) SDS-Tricine gel of BD-CS5 coexpressed with NisBTC or NisT before (&) and after (#) streptavidin column purification. (b) Structure of BD-CS5 with a cyclic strep ligand. (c) Whole-cell ELISA on L. lactis NZ9000 cells demonstrates that cyclic HPQF is displayed on the cell surface; each column represents the mean ± SD of three independent experiments; the statistical significance of differences was evaluated by Pearson r 2 , ns, p > 0.05; * p < 0.05 vs L. lactis NZ9000 cells (without plasmid).

Article Snippet: Aliquots of OD 600 = 3.0 from these cell suspensions were incubated with a 4000-fold diluted horseradish peroxidase (HRP)-conjugated streptavidin solution (Thermo Fisher Scientific, Waltham, MA) in a final volume of 1 mL of MES-Ca plus 0.2% Tween 20 and 1% BSA at room temperature for 1 h under rotation.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: ACS Synthetic Biology

Article Title: High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB

doi: 10.1021/acssynbio.0c00130

Figure Lengend Snippet: Schematic diagram of selection mutant NisB with improved dehydration capacity. Cyclic strep ligand has higher affinity to streptavidin than linear strep ligand, allowing the use of streptavidin-coupled magnetic beads to fish out the bacteria with NisB mut that can dehydrate Ser preceded by Asp.

Article Snippet: Aliquots of OD 600 = 3.0 from these cell suspensions were incubated with a 4000-fold diluted horseradish peroxidase (HRP)-conjugated streptavidin solution (Thermo Fisher Scientific, Waltham, MA) in a final volume of 1 mL of MES-Ca plus 0.2% Tween 20 and 1% BSA at room temperature for 1 h under rotation.

Techniques: Selection, Mutagenesis, Magnetic Beads, Bacteria

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix and Induces Vascular Permeability

doi: 10.1038/labinvest.2014.62

Figure Lengend Snippet: GZMB releases VEGF from human plasma FN. VEGF (50 ng/ml) was added to FN coated wells and incubated for 2 h at 37 °C. Unbound VEGF was removed by washing with DPBS. GZMB (50 nM) with vehicle control (DMSO) or GZMB with inhibitor (Compound 20; 50 μM) were added to the wells and incubated for 2 h at 37°C. Supernatants were removed and analyzed for VEGF by ELISA. Results are presented as mean ± SEM of n=3 with 3 triplicates per condition (*=P<0.05; ** =P<0.01).

Article Snippet: Membranes were blocked with 2.5% milk and immunoblotted using anti-human FN Ab (R&D systems, Minneapolis, MN) at 1:1000 dilution in blocking solution or biotinylated anti-human VEGF (R&D systems, Minneapolis, MN) at 1:100 dilution over night at 4°C.

Techniques: Clinical Proteomics, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix and Induces Vascular Permeability

doi: 10.1038/labinvest.2014.62

Figure Lengend Snippet: GZMB releases VEGF from HUVEC matrix. HUVEC were grown to confluence and maintained in serum-reduced media for 9 d. Cells were removed by adding NH 4 OH followed by extensive washing. Remaining ECM was incubated with VEGF (50 ng/ml) for 2 h at 37°C. Unbound VEGF was removed by washing with DPBS. GZMB (50 nM) or GZMB with inhibitor (Compound 20, 50 μM) were added and incubated for additional 2 h at 37°C. Supernatants were removed and analyzed for VEGF by ELISA. Results are presented as mean ± SEM of n=3 with 3 triplicates per condition (*P<0.05; ** P<0.01).

Article Snippet: Membranes were blocked with 2.5% milk and immunoblotted using anti-human FN Ab (R&D systems, Minneapolis, MN) at 1:1000 dilution in blocking solution or biotinylated anti-human VEGF (R&D systems, Minneapolis, MN) at 1:100 dilution over night at 4°C.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix and Induces Vascular Permeability

doi: 10.1038/labinvest.2014.62

Figure Lengend Snippet: GZMB degrades FN but does not cleave VEGF. A . FN coated culture wells were treated with either GZMB or plasmin (50nM) with or without inhibitors (A, aprotinin) for 2 h at 37 °C. Supernatants were analyzed by western blotting using anti-FN Ab. B. Same enzyme preparations used in (A) were incubated with 100ng VEGF in microtubes for 2 h at 37 °C. Samples were analyzed by western blotting using anti-VEGF Ab (arrow denotes VEGF fragment).

Article Snippet: Membranes were blocked with 2.5% milk and immunoblotted using anti-human FN Ab (R&D systems, Minneapolis, MN) at 1:1000 dilution in blocking solution or biotinylated anti-human VEGF (R&D systems, Minneapolis, MN) at 1:100 dilution over night at 4°C.

Techniques: Western Blot, Incubation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix and Induces Vascular Permeability

doi: 10.1038/labinvest.2014.62

Figure Lengend Snippet: GZMB-mediated VEGF release from FN activates VEGFR2 in HUVEC. FN coated wells were incubated with VEGF and treated with GZMB or GZMB with inhibitor as in . Supernatants were removed and added to HUVEC monolayer culture for 7 min. Cell lysates were analyzed by western blotting using A. phospho y1214 VEGFR2 Ab (pVEGFR2 y1214) and B. phospho y1175 VEGFR2 Ab (pVEGFR2 y1175). HUVEC treated directly with VEGF (50ng/ml) were used as a positive control (+ve ctr). Total VEGFR2 and β-tubulin Abs were used as loading controls. Quantification is presented as the densitometry ratio of pVEGFR2 to total VEGFR2 normalized to β-tubulin. Results are presented as mean ± SEM of n=3 with 3 triplicates per condition (*=P<0.05).

Article Snippet: Membranes were blocked with 2.5% milk and immunoblotted using anti-human FN Ab (R&D systems, Minneapolis, MN) at 1:1000 dilution in blocking solution or biotinylated anti-human VEGF (R&D systems, Minneapolis, MN) at 1:100 dilution over night at 4°C.

Techniques: Incubation, Western Blot, Positive Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix and Induces Vascular Permeability

doi: 10.1038/labinvest.2014.62

Figure Lengend Snippet: Mouse Gzmb induces a VEGF-dependent increase in vascular permeability in vivo . Evan’s blue (0.5%) was injected via the tail vein prior to ear injections of A. mGzmb (100 ng) or saline control or B. mGzmb with anti-mouse VEGF Ab (1.5 μg) or mGzmb and IgG control Ab (1.5 μg). Ear tissues were excised as indicated using a 7 mm punch biopsy (areas in circle). Evan’s blue extraction was performed by drying the tissue and immersing it in formamide at 55°C for 24 h. Results are presented as absorbance (610nm) normalized to tissue weight (mg). Results are presented as mean ± SEM of n=5 (*=P<0.05).

Article Snippet: Membranes were blocked with 2.5% milk and immunoblotted using anti-human FN Ab (R&D systems, Minneapolis, MN) at 1:1000 dilution in blocking solution or biotinylated anti-human VEGF (R&D systems, Minneapolis, MN) at 1:100 dilution over night at 4°C.

Techniques: Permeability, In Vivo, Injection, Saline, Control, Extraction

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix and Induces Vascular Permeability

doi: 10.1038/labinvest.2014.62

Figure Lengend Snippet: Endogenous VEGF expression in mouse ear and mGzmb cleavage of murine FN (mFN). A. Untreated mouse ears were analyzed for endogenous mouse VEGF expression using immunohistochemistry with anti-mouse VEGF Ab (i). Mouse kidney sections were used as positive control for VEGF staining (iii). Ear section (ii) or kidney section (iv) stained without primary antibody were used as negative controls. Scale bar= 100μm. B. Double immunofluorescence using anti-mouse VEGF Ab (i, red) and anti-FN Ab (ii, green) demonstrating co-localization of VEGF and FN (iii, overlay, arrows) in the deep dermis. Scale bar = 47 μm. C. Culture wells coated with mFN were treated with either mGzmb or human GZMB (hGZMB) and incubated for 2 h at 37°C. Supernatants were removed and analyzed by western blotting using anti-FN Ab.

Article Snippet: Membranes were blocked with 2.5% milk and immunoblotted using anti-human FN Ab (R&D systems, Minneapolis, MN) at 1:1000 dilution in blocking solution or biotinylated anti-human VEGF (R&D systems, Minneapolis, MN) at 1:100 dilution over night at 4°C.

Techniques: Expressing, Immunohistochemistry, Positive Control, Staining, Immunofluorescence, Incubation, Western Blot

Journal: Nutrition & Diabetes

Article Title: In utero exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate promotes local adipose and systemic inflammation in adult male offspring

doi: 10.1038/nutd.2014.13

Figure Lengend Snippet: Effect of in utero DEHP exposure on male offspring hepatic and adipose tissue inflammatory marker expression and adipose tissue macrophage infiltration. ( a ) Total adipose tissue quantitative reverse transcription-PCR (qRT-PCR) products of Crp , Il6 and Tnf normalized to Actb. Data are presented as fold over control ( n =7). ( b ) Liver qRT-PCR products of Crp normalized to Actb and presented as fold over control ( n =7). ( c ) Epididymal adipose tissue content of CRP, IL-6 and TNF as determined using enzyme-linked immunosorbent assay (ELISA; n =7, except the 50 mg DEHP group where n =5). ( d ) Representative histological sections of adipose tissue controls and treated with 300 mg DEHP per kg per day stained for CD163 (green) and DAPI (blue). Objective × 20. ( e ) Adipose tissue content of CD163 as determined by ELISA ( n =5 except 300 mg DEHP group where n =6). ( f ) Retroperitoneal adipose tissue content of IL-6 as determined by ELISA ( n =8, except the 1 mg DEHP group where n =4). Results are expressed as the mean±s.e.m., one-way analysis of variance (ANOVA) followed by Dunnett's post hoc tests (*) or t -tests ( # ) were used to calculate statistical significance compared with control; #, * P <0.05. Bars indicate doses significant by ANOVA; * P <0.05.

Article Snippet: Tissue slides were incubated overnight in primary mouse IgG1 anti-CD163 monoclonal antibody solution (1:100 dilution; Acris Antibodies Inc., San Diego, CA, USA).

Techniques: In Utero, Marker, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining