Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet: Serum levels of total and HDM-specific immunoglobulins show a time-of-day response and sex-based differences to chronic HDM exposure Total IgE, Total IgG, HDM-specific IgE, HDM-specific IgG, HDM-specific IgG1, HDM-specific IgG2b, HDM-specific IgM, and HDM-specific IgA in the serum of chronic PBS- and HDM-exposed females and males at ZT0 and ZT12 were determined by ELISA. Data were expressed as ng/ml and mg/ml for total IgE and total IgG, respectively. HDM-specific immunoglobulins (IgE, IgG, IgG1, IgG2b, IgM, and IgA) in the serum were determined by commercially available ELISA kits (Chondrex, Inc.). Unable to detect HDM-specific IgG2a and IgG3 levels in PBS- and HDM-exposed mice. Data for the HDM-specific immunoglobulins were expressed as absorbance at 450 nm or μg/ml. Data are shown as mean ± SEM, Two-way ANOVA followed by Tukey’s multiple comparison test (n = 5/group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared to respective control (PBS) at ZT0 or ZT12; # p < 0.05, compared to HDM at ZT0 vs. ZT12; # # p < 0.01, compared to HDM at ZT0 vs. ZT12. Summary statistics for interaction between Treatment x Sex x Time were analyzed using generalized linear modeling using R (see Table S2 ).

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: iScience

Article Title: Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption

doi: 10.1016/j.isci.2023.107580

Figure Lengend Snippet:

Article Snippet: Mouse Anti-House Dust Mite (HDM) IgG2b Antibody Assay Kit , Chondrex, Inc. , Cat# 3035.

Techniques: Purification, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Expression levels of ErbB2 and ErbB3 on cell lines

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Affinity and binding kinetics of the anti-ErbB3 A5 scFv. k on and k off rates were determined by surface plasmon resonance and used to determine the binding affinity ( K D ) of the A5 scFv. ( A ) Sensorgram fit to 1 : 1 Langmuir binding model. ( B ) Analysis of data.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Binding Assay, SPR Assay

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The anti-ErbB2/ErbB3 bs-scFv ALM. ( A ) Cartoon of ALM depicting scFv orientation, linker sequence and kinetic constants of ALM for each target antigen. ( B ) UV adsorption spectrum chromatograph of ALM over Superdex 75 size-exclusion column.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Sequencing, Adsorption

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: ALM selectively targets ErbB2/ErbB3 positive cells in vitro

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: In Vitro

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The A5-linker-ML3.9 bs-scFv selectively binds BT-474 tumour cells in vitro . Non-labelled BT-474 (ErbB2‘+’/ErbB3‘+’) breast tumour cells were mixed with either an equal ( A and B ) or 18-fold excess ( C ) of fluorescently labelled MCF10a (ErbB2‘±’/ErbB3‘±’) normal breast epithelial cells. Cell mixtures were then incubated with buffer ( A ) or 100 n M ALM ( B and C ) and binding of ALM to each cell population was determined by flow cytometry with an anti-6XHis tag secondary antibody. MCF10a cells were sorted to the upper quadrants and the non-labelled BT-474 cells were sorted to the lower quadrants. Cells bound by the secondary antibody sorted to the respective right hand quadrants. Images on the left depict the raw flow cytometry data. Values on the right represent the absolute number and overall percentage of each cell type in the respective quadrants.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: In Vitro, Incubation, Binding Assay, Flow Cytometry

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Bispecific binding is required for optimal tumour targeting of the ALM bs-scFv in vivo . The biodistributions of radioiodinated ALM, ALD and DLM bs-scFv were analysed 24 h post-injection into xenograft-bearing SCID mice ( n =5 per cohort). ( A ) Co-expression of ErbB2 and ErbB3 by the targeted tumour is required for optimal targeting of ALM in vivo . 125 I-ALM targeted ErbB2+/ErbB3+ tumour xenografts to ⩾3-fold higher levels than xenografts that express only one of the target antigens. ( B ) Radioiodinated ALM ( 125 I-ALM), which is capable of bivalent association with the surface of Sk-OV-3 tumour cells, exhibited increased targeting as compared with ALD and DLM that targeted the tumours monovalently. Error bars represent the standard error of the mean (s.e.m.).

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Binding Assay, In Vivo, Injection, Expressing

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The A5-linker-ML3.9 bs-scFv has intrinsic anti-tumour cell activity. ( A ) Treatment of BT-474 and MDA-361/DYT2 cells with ALM inhibits colony formation in clonogenicity assays. Treatment of ( B ) BT-474 or ( C ) MDA-361/DYT2 cells with A5 scFv, ML3.9 scFv or the combination of both indicates that the majority of the intrinsic anti-tumour cell activity of ALM is due to the anti-ErbB3 A5 scFv arm. Colonies larger than 0.35 m M were counted using an automatic colony counter. Error bars represent the standard deviation.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Activity Assay, Standard Deviation

Journal: iScience

Article Title: The Taspase1/Myosin1f-axis regulates filopodia dynamics

doi: 10.1016/j.isci.2022.104355

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-RNA Polymerase II/POLR2A , Novus Biologicals , Cat#: NB200-598; RRID: AB_2167465.

Techniques: Recombinant, Western Blot, Migration, Isolation, Amplification, Plasmid Preparation, Software

Journal: International archives of allergy and immunology

Article Title: Characterization of Siglec-8 expression on lavage cells after segmental lung allergen challenge

doi: 10.1159/000488951

Figure Lengend Snippet: Gating strategy for 3-color flow cytometry and Siglec-8 expression on BAL eosinophils, basophils, monocytes, and macrophages. Populations in a whole BAL cell preparation obtained 48 h after segmental lung allergen challenge were first gated based on side versus forward scatter (top dot plot) with gate A (high side scatter, low forward scatter) encompassing eosinophils, gate B (low side and forward scatter) including lymphocytes and basophils, gate C (intermediate side and forward scatter) encompassing monocytes, and gate D (high side and forward scatter) encompassing alveolar macrophages. Comparison to plots with side scatter versus CD14/CD16 and CD14 versus CD45, as well as expression or not of CCR3 (not shown), confirmed the locations of these populations. Row 1: Cells from gate A were gated further based on CD14/16 to include eosinophils (CD14/CD16-negative but autofluorescent) and exclude monocytes, macrophages, and possibly neutrophils (CD14/CD16-positive). Row 2: Cells from gate B were gated further based on CD45 to include basophils (CD45-intermediate) and exclude lymphocytes (CD45-bright) and other events (CD45-negative). Row 3: Cells from gate C were gated further based on CD14/CD16 to include monocytes (CD14-high) and exclude lymphocytes and eosinophils (CD14/CD16-negative). Row 4: Cells from gate D were gated further based on CD14/CD16 to include macrophages (CD14-bright). For each row: Left plots, AF647-conjugated isotype control versus CD14/CD16 or CD45; middle plot, Siglec-8 (AF647-conjugated) versus CD14/CD16 or CD45, blue = Siglec-8, red = isotype control; histograms (to the right) of the gated populations in the middle plots, blue = Siglec-8, red = isotype control. Siglec-8 expression shown in this figure is representative of data from 3 subjects.

Article Snippet: PE-conjugated anti-Siglec-8 clone 837535 was from R&D Systems (Minneapolis, MN, USA).

Techniques: Flow Cytometry, Expressing, Comparison

Journal: International archives of allergy and immunology

Article Title: Characterization of Siglec-8 expression on lavage cells after segmental lung allergen challenge

doi: 10.1159/000488951

Figure Lengend Snippet: Gating strategy for 7-color flow cytometry. Populations in a whole BAL cell preparation obtained 48 h after segmental lung allergen challenge and lysed whole blood were stained with a panel of antibodies including a CD3/CD16/CD19/CD56-FITC cocktail, CD45-BV510, HLA-DR-BV421, CD14-BUV395, CD123-PE-Cy7, FcεRI-PE, and Siglec-8-AF647. CD45-bright leukocytes were distinguished from non-cellular debris. Within the CD45-bright region, three populations were identified as CD14-bright (A, light blue region encompassing monocytes and alveolar macrophages), autofluorescent (B, red region encompassing eosinophils and neutrophils), and CD14-negative (C, dark blue region encompassing basophils, dendritic cells, lymphocytes). The CD14-positive cells in region A were further defined as CD14-very bright and HLA-DR-positive monocytes (light blue region) and CD14-moderate and HLA-DR-positive alveolar macrophages (orange region). Contaminating T cells, neutrophils, B cells, and natural killer cells were eliminated using CD3/CD16/CD19/CD56-staining with forward side scatter (FSC). Doublets were removed based on forward scatter height (FSC-H) versus area (FSC-A). The autofluorescent cells in the BUV395 channel within region B were distinguished using CD3/CD16/CD19/CD56 and side scatter (SSC). Eosinophils (red region) were autofluorescent in the FITC channel with high SSC and neutrophils (purple region), which express CD16, were identified within the CD3/CD16/CD19/CD56-bright population as having moderate SSC. Doublets were eliminated using FSC-H versus FSC-A. Single cells within the CD14-negative cells in region C were identified using FSC-H versus FSC-A and then forward scatter width (FSC-W) versus area (FSC-A). Basophils were defined as CD123-bright and FcεRI-bright, while plasmacytoid dendritic cells (pDCs) were identified as CD123-bright and FcεRI-moderate. pDCs also expressed HLA-DR (not shown). A color overlay displaying contour plots of each cell population confirms appropriate FSC and SSC properties. This 7-color flow cytometry method was used for samples from 3 subjects and those subjects were different from those used in Figures 1 and ​and33.

Article Snippet: PE-conjugated anti-Siglec-8 clone 837535 was from R&D Systems (Minneapolis, MN, USA).

Techniques: Flow Cytometry, Staining

Journal: International archives of allergy and immunology

Article Title: Characterization of Siglec-8 expression on lavage cells after segmental lung allergen challenge

doi: 10.1159/000488951

Figure Lengend Snippet: Quantitation of Siglec-8 expression on BAL eosinophils, monocytes, macrophages, and basophils. Populations of BAL cells obtained 48 h after segmental lung allergen challenges were gated as in Figure 1. Siglec-8 expression presented as specific geometric channel fluorescence (gMCF), mean ± standard deviation (SD), n = 3 subjects.

Article Snippet: PE-conjugated anti-Siglec-8 clone 837535 was from R&D Systems (Minneapolis, MN, USA).

Techniques: Quantitation Assay, Expressing, Fluorescence, Standard Deviation

Journal: International archives of allergy and immunology

Article Title: Characterization of Siglec-8 expression on lavage cells after segmental lung allergen challenge

doi: 10.1159/000488951

Figure Lengend Snippet: Confirmation of Siglec-8 expression on cell subsets using 7-color flow cytometry. Populations of BAL and blood cells obtained 48 h after segmental lung allergen challenge were gated as in Figure 2. Histograms show Siglec-8 expression on each gated population within the fully-stained sample (blue) compared to the comparable population within the fluorescence minus one (FMO) control sample containing all antibodies minus the Siglec-8-AF647 antibody (red). Data are representative of experiments with 3 subjects who were different from the subjects used in Figures 1 and ​and33.

Article Snippet: PE-conjugated anti-Siglec-8 clone 837535 was from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence

Journal: International archives of allergy and immunology

Article Title: Characterization of Siglec-8 expression on lavage cells after segmental lung allergen challenge

doi: 10.1159/000488951

Figure Lengend Snippet: Localization of Siglec-8 on blood and BAL eosinophils. Localization of Siglec-8 (green) and nucleus (blue) in cytospun unactivated blood eosinophils (A), IL-5-activated blood eosinophils (IL-5, 50 ng/ml for 10 minutes as described in Materials and Methods) (B), and BAL eosinophils (C). Cells were analyzed by immunofluorescent staining using mAb to Siglec-8 and AF488-conjugated secondary antibody. Nuclei were stained with DAPI. Bar, 10 µm. Representative of two experiments (i.e., 2 subjects, these 2 subjects were different from the 6 subjects used in Figures 1–4). (D) Quantitation of Siglec-8 localization using Fiji software, peripheral Siglec-8 staining as percentage of circumference in unactivated blood eosinophils (blood eos), activated blood eosinophils (blood eos+IL5), and BAL eosinophils (BAL eos), *p (probability) < 0.05, ***p < 0.001 versus unactivated blood eosinophils.

Article Snippet: PE-conjugated anti-Siglec-8 clone 837535 was from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Quantitation Assay, Software

Journal: Biochemistry

Article Title: Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein

doi: 10.1021/acs.biochem.7b00700

Figure Lengend Snippet: Comparison of wild-type (wt) TIMP-2 (pT2MO1 coding sequence) and codon-optimized (CO) rhTIMP-2-6XHis (COT2his) cDNA and protein sequences. (A) ClustalW (MacVector version 15.5.0) pairwise nucleotide alignment and comparison of the wt TIMP-2 (pT2MO1) and codon-optimized rhTIMP-2-6XHis (COT2his) cDNA sequences using a 10-nucleotide window indicating changes made to and from G and C nucleotides. (B) Comparison of the % GC content in the pT2MO1 (blue) and COT2his (red) lines in 10-nucleotide sequence windows using the MacVector software. (C) Alignment of the translated protein sequences from wt (pT2MO1) and codon-optmized rhTIMP-2-6XHis (COT2his) cDNA sequences.

Article Snippet: Separated proteins were blotted and probed with the murine anti-TIMP-2 antibody (catalog no. MAB 9711, R&D Systems) and anti-His-6 (catalog no. MA1–21315, Pierce).

Techniques: Sequencing, Software

Journal: Biochemistry

Article Title: Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein

doi: 10.1021/acs.biochem.7b00700

Figure Lengend Snippet: Analysis of two-step downstream purification of rhTIMP-2– 6XHis following bioprocess scale expression. Samples from the bioprocess purification were analyzed as shown in the (A) PageBlue Protein-stained SDS−PAGE gel of the fractions obtained from the IMAC (HisTrap column) purification step. Lane M contained the molecular weight standards (SeeBlue Plus 2 Prestained Standards, Invitrogen, catalog no. LC 5925). Lane CM contained the starting condition medium sample obtained from the HEK-293-F suspension culture. Lane FT contained the flow-through (unbound) fraction obtained during sample loading. Lane NSE contained the nonspecific elution obtained during step gradient elution with 20 mM imidazole. Lane SE contained the specific elution fraction obtained following 250 mM imidazole step elution. (B) PageBlue stained SDS−PAGE gel showing that the rhTIMP-2-6XHis-containing fractions from the IMAC (HisTrap) purification contain a predominant 22 kDa band with minor higher-molecular weight contaminants. The reverse phase HPLC purification effectively removed these higher-molecular weight contaminants, resulting in a single 22 kDa band with >95% purity as estimated by densitometry using a Bio-Rad ChemiDoc XRS+ instrument with Image Lab software. (C) Western Blot analysis of the IMAC fractions using anti-TIMP-2 (top) anti-6XHis tag (bottom) antibodies. The lanes are labeled the same as in panel A.

Article Snippet: Separated proteins were blotted and probed with the murine anti-TIMP-2 antibody (catalog no. MAB 9711, R&D Systems) and anti-His-6 (catalog no. MA1–21315, Pierce).

Techniques: Purification, Expressing, Staining, SDS Page, Molecular Weight, Software, Western Blot, Labeling

Journal: Biochemistry

Article Title: Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein

doi: 10.1021/acs.biochem.7b00700

Figure Lengend Snippet: Inhibition of MMP-2 activity by rhTIMP-2-6XHis and Ala +TIMP-2 was monitored by colorimetric product formation at 412 nm. (A) MMP-2 enzymatic activity data plotted vs log molar concentration of rhTIMP-2-6XHis (red) and Ala+TIMP-2 (blue) were fitted to a nonlinear curve for the one-site enzyme inhibitor response. (B) Data from the linear range of the rhTIMP-2-6XHis inhibition curve were analyzed by linear regression analysis and extrapolation to the X-axis to estimate the molar ratio of rhTIMP-2-6XHis to MMP-2, resulting in complete inhibition of MMP-2 enzymatic activity (n = 3; mean ± standard error of the mean).

Article Snippet: Separated proteins were blotted and probed with the murine anti-TIMP-2 antibody (catalog no. MAB 9711, R&D Systems) and anti-His-6 (catalog no. MA1–21315, Pierce).

Techniques: Inhibition, Activity Assay, Concentration Assay

Journal: Cell Reports

Article Title: Stromal Cell-Contact Dependent PI3K and APRIL Induced NF-κB Signaling Prevent Mitochondrial- and ER Stress Induced Death of Memory Plasma Cells

doi: 10.1016/j.celrep.2020.107982

Figure Lengend Snippet:

Article Snippet: Anti-mouse BCL2, APC, REA356 , Miltenyi Biotec , Catalog # 130-105-474; RRID: AB_2651266.

Techniques: Recombinant, Electron Microscopy, Staining, Software