Journal: Membranes

Article Title: The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

doi: 10.3390/membranes11030218

Figure Lengend Snippet: Specific binding of anti-T-cadherin antibodies to megakaryocytes MEG-01 and platelets compared with negative THP-1 control and positive human umbilical vein endothelial cells (HUVEC) control according to flow cytometry data. Forward scatter /Side scatter gates were cell specific and channel voltage for detection of T-cadherin (ex640 em670/14) was the same for all cells (THP-1, HUVEC, MEG-01) and different for platelets. ( a ) Distribution histograms of cells incubated with antibodies anti-T-cadherin (blue) or isotypic control (red). ( b ) Median and percentiles of the fluorescence (antibody binding) for the above histograms. * The binding of the specific antibody is greater than of the isotype control, p < 0.001. n = 5. ( c ) Median and percentiles of the fluorescence (antibody binding) for blank sample without antibody (Bl), sample with rabbit IgG (IG_R), ProSci (PS), SantaCruz (SC), and Abnova (Ab) antibodies. * The binding of the specific antibody is greater than of the isotype control, p < 0.001. n = 2.

Article Snippet: Briefly, we used Dynabeads M-280 Streptavidin (Invitrogen) and biotinylated antibody anti-T-cadherin (R&D #BAF3264).

Techniques: Binding Assay, Flow Cytometry, Incubation, Fluorescence

Journal: Membranes

Article Title: The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

doi: 10.3390/membranes11030218

Figure Lengend Snippet: Validation of antibody #BAF3264. Fixed cells. Hoechst 33342 (DNA)—blue, #BAF3264 (T-cadherin)—red: ( a ) Wild-type CHO; ( b ) CHO cells with T-cadherin expression. Scale bar (red line) 10 µm.

Article Snippet: Briefly, we used Dynabeads M-280 Streptavidin (Invitrogen) and biotinylated antibody anti-T-cadherin (R&D #BAF3264).

Techniques: Expressing

Journal: Membranes

Article Title: The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

doi: 10.3390/membranes11030218

Figure Lengend Snippet: Representative confocal microscopy image (a single focal plane) of T-cadherin on platelets. CD61—green, T-cadherin—red, DNA (Hoecst 33342)—blue. Live platelet and nucleated blood cells. Scale bar (white line) 2 µm.

Article Snippet: Briefly, we used Dynabeads M-280 Streptavidin (Invitrogen) and biotinylated antibody anti-T-cadherin (R&D #BAF3264).

Techniques: Confocal Microscopy

Journal: Membranes

Article Title: The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

doi: 10.3390/membranes11030218

Figure Lengend Snippet: Digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) decreases binding of the specific antibody against T-cadherin to platelets ( a ) and MEG-01 ( b ). Platelets are identified by their FSC/SSC characteristic (the first gate) and positive CD61 labeling (the second gate), MEG-01 cells are identified by the other FSC/SSC gate. Right histograms show fluorescence from the binding of the anti-T-cadherin antibody before (blue) and after (red) PI-PLC treatment and from the isotypic control (gray).

Article Snippet: Briefly, we used Dynabeads M-280 Streptavidin (Invitrogen) and biotinylated antibody anti-T-cadherin (R&D #BAF3264).

Techniques: Binding Assay, Labeling, Fluorescence

Journal: Membranes

Article Title: The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

doi: 10.3390/membranes11030218

Figure Lengend Snippet: Representative immunoblots against T-cadherin with platelet and positive controls lysates with protein bands standards, n = 5. ( a ) First three lanes were incubated with R&D #AF3264 antibodies, last three lanes were incubated with ProSci #3583 antibodies, H–HUVEC, pl1, pl2—platelets from different donors, HT–HEK293 overexpressing T-cadherin (preparation describe in ) (film), ( b ) H–HUVEC, pl—immunoprecipitate of the platelet lysate (R&D #AF3264, multichannel digital detection).

Article Snippet: Briefly, we used Dynabeads M-280 Streptavidin (Invitrogen) and biotinylated antibody anti-T-cadherin (R&D #BAF3264).

Techniques: Western Blot, Incubation

Journal: Membranes

Article Title: The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

doi: 10.3390/membranes11030218

Figure Lengend Snippet: Analysis of fractions after ultracentrifugation of platelet lysates with the Triton X-114 detergent. 1–12—the fractions obtained after ultracentrifugation. Upper panel—immunoblotting of the obtained fractions with antibodies against T-cadherin. The middle panel shows the total amount of protein in the fractions, analyzed using amide black. The lower panel shows the ratio of the T-cadherin signal intensity measured by immunoblotting to the total protein amount in the fractions, arbitrary units.

Article Snippet: Briefly, we used Dynabeads M-280 Streptavidin (Invitrogen) and biotinylated antibody anti-T-cadherin (R&D #BAF3264).

Techniques: Western Blot

Journal: Membranes

Article Title: The Missing Protein: Is T-Cadherin a Previously Unknown GPI-Anchored Receptor on Platelets?

doi: 10.3390/membranes11030218

Figure Lengend Snippet: The confocal microscopy images and orthogonal view of Z-stack of resting and activated platelets. CD61—green, T-cadherin—red. Plots of fluorescence intensity along the cross-section line from ( a , b ), respectively. ( a ) Fixed resting human platelets—non-permeabilized (left) and permeabilized (right); ( b ) fixed and permeabilized activated human platelet; ( c ) live activated human platelet. Scale bar (white line) 2 µm.

Article Snippet: Briefly, we used Dynabeads M-280 Streptavidin (Invitrogen) and biotinylated antibody anti-T-cadherin (R&D #BAF3264).

Techniques: Confocal Microscopy, Fluorescence

Journal: Biotechnology and bioengineering

Article Title: Human Trabecular Meshwork Cell Behavior is Influenced by Collagen Scaffold Pore Architecture and Glycosaminoglycan Composition

doi: 10.1002/bit.27477

Figure Lengend Snippet: The fibronectin expression quantified on collagen‐only scaffolds using qPCR. Results are shown as a relative fold change by the ΔΔCt method. (*p < .05 and ***p < .005). AFF, aligned flash frozen; NAFF, nonaligned flash frozen; qPCR, qualitative polymerase chain reaction.

Article Snippet: Anti-Fibronectin antibody (Biotin) (ab6584) was obtained from Abcam (Cambridge, UK).

Techniques: Expressing, Polymerase Chain Reaction

Journal: Biotechnology and bioengineering

Article Title: Human Trabecular Meshwork Cell Behavior is Influenced by Collagen Scaffold Pore Architecture and Glycosaminoglycan Composition

doi: 10.1002/bit.27477

Figure Lengend Snippet: Confocal micrographs of collagen‐only scaffolds after week 2 of culture. Fibronectin (red) and nuclei (blue). AFF, aligned flash frozen; CO, collagen only; NAFF, nonaligned flash frozen.

Article Snippet: Anti-Fibronectin antibody (Biotin) (ab6584) was obtained from Abcam (Cambridge, UK).

Techniques:

Journal: Biotechnology and bioengineering

Article Title: Human Trabecular Meshwork Cell Behavior is Influenced by Collagen Scaffold Pore Architecture and Glycosaminoglycan Composition

doi: 10.1002/bit.27477

Figure Lengend Snippet: The fibronectin expression quantified by qPCR on all scaffolds and grouped by the scaffold type. All data are normalized to NAFF–CO at their respective timepoints. (*p < .05). CO, collagen only; CSHA, chondriotin sulfate hyaluronic acid; qPCR, quantitative polymerase chain reaction.

Article Snippet: Anti-Fibronectin antibody (Biotin) (ab6584) was obtained from Abcam (Cambridge, UK).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Biotechnology and bioengineering

Article Title: Human Trabecular Meshwork Cell Behavior is Influenced by Collagen Scaffold Pore Architecture and Glycosaminoglycan Composition

doi: 10.1002/bit.27477

Figure Lengend Snippet: Confocal micrographs of CSHA scaffolds after 2 weeks of culture. Fibronectin (red) and nuclei (blue). AFF, aligned flash frozen; CSHA, chondriotin sulfate hyaluronic acid; NAFF, nonaligned flash frozen.

Article Snippet: Anti-Fibronectin antibody (Biotin) (ab6584) was obtained from Abcam (Cambridge, UK).

Techniques:

Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: PBMCs were stimulated for 24 hr with CpG/ionomycin/PMA and BFA as described previously and analyzed by FACS for annexin V (AnV) binding and IL-10 and TNFα production in 21 subjects. Cytometry gating is shown in A. Results are presented in percentage of positive cells in B and median of fluorescence (MFI) in C. Wilcoxon’s matched pairs signed rank tests were used. Top of the bar represents the median, and whiskers are IQR 25–75.

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques: Binding Assay, Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: A representative plot is presented (A). PBMCs from 12 subjects were analyzed by FACS for B10 precursors phenotype (i.e CD19+CD5+ (B), CD19+CD24 hi CD27 + (C) and CD19+CD24 hi CD38 hi , (D)), and for annexin V binding. Wilcoxon’s matched pairs signed rank tests were used. Data are median (IQR 25–75).

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: PBMCs from 6 healthy subjects were sorted by flow cytometry (FACSARIA) according to annexin V (AnV) staining: AnV positive (AnV + ) and AnV negative B cells (AnV neg ) after exclusion of dead cells (DAPI + )(A). Each B cell subpopulation was then stimulated for B10 generation and analyzed by flow cytometry as previously described (B). (C) shows the % of cytokine-positive cells (IL-10, IL-6, GM-CSF and TNF-a) in AnV + and AnV AnV neg sorted cells. (D) shows IL-10 and IL-6 concentrations assessed by ELISA in supernatant of AnV + and AnV AnV neg sorted cells.

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques: Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: PBMCs were stained with APC anti-CD19 and FITC conjugated Annexin V, sorted by FACSARIA according to annexin V (AnV) staining: high AnV B cells (AnV hi ); low AnV B cells (AnV low ) and AnV negative B cells (AnV neg ). Cell death was analyzed at 72 hr using DAPI staining for necrosis (A) and DIOC6 (B) for mitochondrial apoptosis (n = 4). Additionally, cell cycle was analyzed using propidium iodide (PI) staining at 72 hr (C), showing the proportion of cells in the subG1 phase (apoptotic cells) and in the S phase (proliferating cells) in AnV+ (AnV hi and AnV low ) and AnV neg B cells (n = 5). Data are median (IQR25-75).

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques: Staining

Journal: PLoS ONE

Article Title: Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

doi: 10.1371/journal.pone.0169755

Figure Lengend Snippet: PBMCs from 6 healthy subjects were pretreated with biotinylated Annexin V (biot AnV) (n = 8) or with glyburide (Gly) (n = 8) and then stimulated for 24 hr according to the protocol for B10 assessment previously described. A shows representative plots and B shows the results as median (IQR25-75).

Article Snippet: Briefly, 2.10 6 of PBMCs were washed, pre-incubated in annexin binding buffer containing 5 μl of biotinylated Annexin V (BD pharmingen), washed and further incubated with streptavidine.

Techniques:

Journal: iScience

Article Title: Extensive three-dimensional intratumor proteomic heterogeneity revealed by multiregion sampling in high-grade serous ovarian tumor specimens

doi: 10.1016/j.isci.2021.102757

Figure Lengend Snippet:

Article Snippet: Goat Anti-rabbit IgG , Vector Laboratories, Inc. , Cat# BA1000.

Techniques: Plasmid Preparation, Recombinant, Protease Inhibitor, Protein Extraction, Staining, Stripping Membranes, Blocking Assay, Avidin-Biotin Assay, Amplification, Bicinchoninic Acid Protein Assay, RNA HS Assay, Expressing, Quantitation Assay, Software