Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Functional K Ca 3.1 channels regulate steroid insensitivity in bronchial smooth muscle cells 1

doi: 10.4049/jimmunol.1300104

Figure Lengend Snippet: Cells pre-treated with TRAM-34 (200 nM) or ICA-17043 (60 nM) for 2 h were stimulated with TNFα/IFNγ for 22 h. PP5 levels assessed by flow cytometry were expressed as the fold increase in mean fluorescence intensity (MFI) over basal ± S.E.M. of experiments performed in 9 subjects (n=4 asthmatics and n=5 healthy controls). *P <0.05; **P < 0.01.

Article Snippet: Cells were then stained with either 2 μg/ml rabbit anti-human PP5 antibody (Cell Signaling, Danvers, MA USA) or isotype matched control (rabbit IgG-Immunostep, Salamanca Spain) overnight, followed by secondary sheep anti-rabbit IgG:RPE antibody (Cell Signalling, Danvers, MA USA) for 2 hr.

Techniques: Flow Cytometry, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Functional K Ca 3.1 channels regulate steroid insensitivity in bronchial smooth muscle cells 1

doi: 10.4049/jimmunol.1300104

Figure Lengend Snippet: We uncovered two mechanisms by which KCa3.1 channels mediate TNFα/IFNγ-associated corticosteroid insensitivity. Transcription of some pro-asthmatic genes involves KCa3.1-dependent pathways that are insensitive to corticosteroids (right). Other genes are resistant to corticosteroids via the induction of the KCa3.1-dependent serine/threonine phosphatase PP5 which impairs GRα transcriptional function through decreased GRα phosphorylation (left).

Article Snippet: Cells were then stained with either 2 μg/ml rabbit anti-human PP5 antibody (Cell Signaling, Danvers, MA USA) or isotype matched control (rabbit IgG-Immunostep, Salamanca Spain) overnight, followed by secondary sheep anti-rabbit IgG:RPE antibody (Cell Signalling, Danvers, MA USA) for 2 hr.

Techniques: Phospho-proteomics

Journal: Shock

Article Title: PROTEASE-ACTIVATED RECEPTOR 2 BLOCKING PEPTIDE COUNTERACTS ENDOTOXIN-INDUCED INFLAMMATION AND COAGULATION AND AMELIORATES RENAL FIBRIN DEPOSITION IN A RAT MODEL OF ACUTE RENAL FAILURE

doi: 10.1097/shk.0b013e3181a5359c

Figure Lengend Snippet: FIG. 1. An immunoblot analysis of tissue factor (A); an immunoblot analysis of TFPI (B); an immunoblot analysis of !-thrombin (C); ELISA of PAI-1 (D); and immunoblot analyses of fibrinogen and fibrin in kidney tissues (E and F). All of the experiments were done in the control rats and at different time points after LPS administration (n = 17). In each of the immunoblot experiments, the band obtained in the control was normalized to 1.0. *P G 0.01 in comparison to the control.

Article Snippet: For immunological detection (immunohistochemistry and immunoblot analysis), the following antibodies were used: antihuman PAR-2 goat polyclonal antibody (Santa Cruz Biotechnology); antihuman fibrin mouse monoclonal antibody (Chemicon International, Temecula, Calif), antihuman thrombin goat polyclonal antibody (R&D Systems, Inc., Minneapolis, Minn), antihuman coagulation FVII goat polyclonal antibody (R&D Systems, Inc.), rabbit polyclonal FX (H-120) antibody (Santa Cruz Biotechnology), monoclonal antihuman FXa antibody (R&D Systems, Inc.), rabbit polyclonal TFPI antibody (Santa Cruz Biotechnology), antirabbit tissue factor sheep polyclonal antibody (American Diagnostica, Stamford, Conn), rabbit antirat PAI-1 antibody (American Diagnostica), murine monoclonal antibody against human FVIIa (American Diagnostica), and antiYXenopus laevis "-actin mouse monoclonal antibody (Abcam, Cambridge, U.K.).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Control, Comparison

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Inhibition of Thrombin Receptor Signaling on α-Smooth Muscle Actin + CD34 + Progenitors Leads to Repair After Murine Immune Vascular Injury

doi: 10.1161/atvbaha.111.239046

Figure Lengend Snippet: Figure 2. Adoptive transfer of CD34 or CD34 cells. Pheno- typic analysis of BALB/c aortas in wild-type (WT) mice following adoptive transfer of 1106 cells 1 week posttransplantation. Cells harvested from mice 2 days postinjury. n5 per group. Aortas were harvested at 6 (A, B, and D) or 2 (C) weeks. Results are representative of 3 independent experiments. A and B, Quantitative analysis of neointimal area (A) and neointima:m- edia ratios (B). Columns show mean valuesSEM. Data points are derived from 3 random sections from 5 different vessels. WT:FIIa indicates WT cells incubated with thrombin; WT:protease-activated receptor-1 (PAR-1) antag; WT cells incu- bated with PAR-1 antagonist; PAR-1/PAR-4 KO, cells from PAR-1- or PAR-4-deficient mice. C and D, Representative 2-color (right) immunofluorescence of sections (green indicates human tissue factor pathway inhibitor [hTFPI] [C] or yellow fluo- rescent protein (YFP) [D]) after injection of cells from -tissue factor pathway inhibitor–transgenic (-TFPI-Tg) (A) or ROSA- enhanced yellow fluorescent protein (EYFP) (B) mice. L indicates lumen; N, neointima; M, media; A, adventitia. Blue indicates 4,6-diamidino-2-phenylindole. Fluorescence from the YFP was captured through a green filter to aid colocalization.

Article Snippet: The Ab used were monoclonal rat anti- mouse CD34, CD68 (Serotec, Oxford, United Kingdom), and CD31, CD3, CD4, CD8 (BD Bioscience Pharmingen, Oxford, United Kingdom); polyclonal rabbit anti–TF, VEGF-R2 (ABR-Affinity BioReagents,Golden, CO), SM22α, P-selectin (BD Bioscience Pharmingen), anti-PAR 1 and anti-PAR 4 (Autogenbioclear, Wiltshire, UK) and human TFPI (American Diagnostica, Stamford, CA, USA); mouse 5 monoclonal anti-PAR 21 (Zymed, California, USA) and anti–human α-SMA (Sigma- Aldrich, St Louis, MO, USA); chicken anti–mouse E-selectin (R&D Systems, Minneapolis, MN, USA); hamster anti-mouse CD11c (Serotec).

Techniques: Adoptive Transfer Assay, Derivative Assay, Incubation, Injection, Transgenic Assay, Fluorescence

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Inhibition of Thrombin Receptor Signaling on α-Smooth Muscle Actin + CD34 + Progenitors Leads to Repair After Murine Immune Vascular Injury

doi: 10.1161/atvbaha.111.239046

Figure Lengend Snippet: Figure 3. Analysis of circulating CD34 cells. A, CD34 circulat- ing in wild-type (WT) mice after allogeneic (squares) or synge- neic transplantation (Tx). *P0.05, †P0.001 (n6 per group). B, Phenotype of circulating CD34 cells after allogeneic (black) or syngeneic (white) transplantation. Each graph shows the pro- portion of CD34 cells expressing tissue factor (TF), protease- activated receptor-1 (PAR-1), or -smooth muscle actin (-SMA). Squares indicate WT; circles, -tissue factor pathway inhibitor–transgenic (-TFPI-Tg). In A and B, day 020 hours postoperation. *P0.05, †P0.01 (n6 per group). C, Circulat- ing CD34 cells, isolated from -TFPI-Tg mice 7 days after BALB/c aortic transplantation, examined by immunocytofluores- cence. Cells were stained with human tissue factor pathway in- hibitor (hTFPI) (red) and CD45 (green). Blue indicates 4,6- diamidino-2-phenylindole. Right panel shows combined image, illustrating expression of hTFPI fusion protein by CD45 cells (yellow arrow) and CD45 cells (red arrows). Results are repre- sentative of 3 independent experiments.

Article Snippet: The Ab used were monoclonal rat anti- mouse CD34, CD68 (Serotec, Oxford, United Kingdom), and CD31, CD3, CD4, CD8 (BD Bioscience Pharmingen, Oxford, United Kingdom); polyclonal rabbit anti–TF, VEGF-R2 (ABR-Affinity BioReagents,Golden, CO), SM22α, P-selectin (BD Bioscience Pharmingen), anti-PAR 1 and anti-PAR 4 (Autogenbioclear, Wiltshire, UK) and human TFPI (American Diagnostica, Stamford, CA, USA); mouse 5 monoclonal anti-PAR 21 (Zymed, California, USA) and anti–human α-SMA (Sigma- Aldrich, St Louis, MO, USA); chicken anti–mouse E-selectin (R&D Systems, Minneapolis, MN, USA); hamster anti-mouse CD11c (Serotec).

Techniques: Transplantation Assay, Expressing, Transgenic Assay, Isolation, Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Inhibition of Thrombin Receptor Signaling on α-Smooth Muscle Actin + CD34 + Progenitors Leads to Repair After Murine Immune Vascular Injury

doi: 10.1161/atvbaha.111.239046

Figure Lengend Snippet: Figure 5. Recruitment of cells to aortas in -tissue factor path- way inhibitor–transgenic (-TFPI-Tg) recipients. A to D, Three- color immunohistological analysis of frozen sections from BALB/c aortas transplanted into -TFPI-Tg mice. Day 020 hours posttransplantation. All sections were stained with 4,6- diamidino-2-phenylindole (blue) and anti-CD31, CD34, CD45, or -smooth muscle actin (-SMA) as indicated (red) (A); or anti- human tissue factor pathway inhibitor (hTFPI) or CD31 as indi- cated (green) (B). C, Overlay images. D, Enlarged images from the boxed areas in C. Yellow indicates colocalization. Colored arrows in images from the same day indicate the same cell in consecutive sections. Results are representative of 3 indepen- dent experiments.

Article Snippet: The Ab used were monoclonal rat anti- mouse CD34, CD68 (Serotec, Oxford, United Kingdom), and CD31, CD3, CD4, CD8 (BD Bioscience Pharmingen, Oxford, United Kingdom); polyclonal rabbit anti–TF, VEGF-R2 (ABR-Affinity BioReagents,Golden, CO), SM22α, P-selectin (BD Bioscience Pharmingen), anti-PAR 1 and anti-PAR 4 (Autogenbioclear, Wiltshire, UK) and human TFPI (American Diagnostica, Stamford, CA, USA); mouse 5 monoclonal anti-PAR 21 (Zymed, California, USA) and anti–human α-SMA (Sigma- Aldrich, St Louis, MO, USA); chicken anti–mouse E-selectin (R&D Systems, Minneapolis, MN, USA); hamster anti-mouse CD11c (Serotec).

Techniques: Transgenic Assay, Staining

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Postinjury vascular intimal hyperplasia in mice is completely inhibited by CD34+ bone marrow-derived progenitor cells expressing membrane-tethered anticoagulant fusion proteins.

doi: 10.1111/j.1538-7836.2006.02100.x

Figure Lengend Snippet: Fig. 2. (A) Two-color analysis of the acute injury in WT mice. Consecu- tive sections were stained with a-smooth muscle actin (SMA) (red in all panels) and (green) anti-CD41 (platelets), antifibrin or anti-CD68 (macr- ophages). Yellow indicates co-localization. Lumen is uppermost in all images. The day 0 image is taken immediately following the injury. White arrows indicate platelet clumps or infiltrating macrophages. Magnification ·100. (B) Two-colour analysis of tissue factor (TF) expression after injury. Consecutive sections were stained with anti-a-SMA (red in all panels) and anti-TF (green). Yellow indicates co-localization. Timing of images as in (A). Magnification ·100.

Article Snippet: Vessels were embedded in OCT (VWR International, Dorset, UK) by freezing with CO2, sectioned and fixed in 100% methanol at )20 C. Frozen sections were immersed in 1% BSA-PBS and 10% goat serum (Sigma) for 30 min, then incubatedovernight at 4 Cwithoneof the followingantibodies (Ab): rabbit antihuman TFPI, sheep anti-Hir (American Diagnostica Inc., Greenwich, CT, USA), mouse antihuman fibrin II b chain Ab (NYB-T2G1, Accurate Chemical Scientific Corp.), rat antimouse CD41 (Pharmingen, San Diego, CA, USA), rat antimouse CD68 (Serotec, Oxford, UK), rabbit anti mouse TF [26] or mouse antihuman a-SMA (Sigma).

Techniques: Staining, Expressing