fab3744p  (R&D Systems)

Journal: Journal of Translational Medicine

Article Title: Defective formyl peptide receptor 2/3 and annexin A1 expressions associated with M2a polarization of blood immune cells in patients with chronic obstructive pulmonary disease

doi: 10.1186/s12967-018-1435-5

Figure Lengend Snippet: Changes in FPR1/2/3 expressions of blood immune cells in COPD patients after 1-year treatment and with various clinical phenotypes. In 10 COPD patients after 1-year treatment, a FPR3 expression of M1 monocyte, b NK cell, and c Th cell, as well as d FPR2 expression on Th cell all showed significant elevation. e COPD patients with a high MMRC dyspnea scale had higher FPR1 expression on neutrophil, while f those with a history of frequent moderate exacerbation in the past 1 year had lower FPR2 expression on neutrophil. g FPR1 expression on neutrophil showed significant reduction after 1-year treatment. h Ex-smokers with COPD had higher FPR3 expression of NK T cell than current smokers with COPD after 1 year treatment. COPD patient receiving oral steroid treatment for more than 3 months had i higher FPR2 expression on Th cell and j higher FPR3 expression of M1 monocyte than those without oral steroid use. *p < 0.05 for comparisons between COPD patients with a specific phenotype and healthy non-smokers by ANOVA test. # p < 0.05 for comparison between COPD patients with and without s specific phenotype by ANOVA test. § p < 0.05 for comparisons between COPD patients with and without a specific management after 1-year follow-up

Article Snippet: (A) Surface markers were measured by simultaneously staining for 30 min at 4 °C with directly conjugated monoclonal antibodies (mAb) as follows: PE Mouse Anti-Human CD56 (BD Pharmingen, USA), PE-CyTM5 Mouse Anti-Human CD3 (BD Pharmingen), CD4-PC7 (Beckman Coulter; USA), CD8-PC7, CD16-PC7, CD14-PC7 (Beckman Coulter), PerCP-Cy5.5 Mouse Anti-Human CD209 (BD Pharmingen), Anti-human CFS-conjugated FPR1 (R&D Systems; USA), PE-conjugated anti-human FPR2 (R&D Systems) or isotype control mAb. (B) For intracellular staining, cells were stained with anti-CD8a-PE-Cyanine5 and incubated with Cytofix/Cytoperm TM for 20 min at 4 °C, and washed with Perm/Wash Buffer.

Techniques: Expressing, Comparison

Journal: Journal of Biological Chemistry

Article Title: The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

doi: 10.1074/jbc.m112.411181

Figure Lengend Snippet: FIGURE 1. Lens epithelial cells constitutively express FPR1; RNA and immunophenotypic evidence. A, RNA is shown. FHL 124 cells nucleofected with the shRNA indicated on the x axis were analyzed for FPR1 mRNA expression by PCR. B, immunophenotype is shown. FHL 124 cells nucleofected with the shRNAs indicated on the x axis were analyzed by FACS with a PE-labeled anti-FPR1 mAb. C, ligand binding; FHL 124 cells nucleofected with the shRNAs indicated on the x axis were analyzed by FACS with the fluorescent agonist ligand fNLFNYK-Fl (10 nM). D, Ca2 assay; FHL 124 cells nucleofected with the shRNAs indicated in the legend were stimulated with 1 M fMLF, and the Ca2 response was followed for 150 s. The base line (first 20 s) of each individual curve and a background curve recorded with only buffer (no fMLF) were subtracted from each signal. Then all experiments were pooled and transformed to % of the peak signal intensity of the siRNA-free (only H2O) control. All data are the means S.E.

Article Snippet: PE-labeled mouse anti-FPR1 mAb (25 g/ml stock solution, catalog no. FAB3744P, clone 350418) and PElabeled IgG2A control (10 g/ml, catalog no. IC003P, clone 20102) were from R&D Systems (Minneapolis, MN).

Techniques: shRNA, Expressing, Labeling, Ligand Binding Assay, Transformation Assay, Control

Journal: Journal of Biological Chemistry

Article Title: The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

doi: 10.1074/jbc.m112.411181

Figure Lengend Snippet: FIGURE 2. Lens epithelial cells constitutively express FPR1. Western blots were probed in panels A and B and the top portion of C with NFPR2, a mAb to FPR1. PMN stands for polymorphonuclear leukocytes (neutrophils). The bottom portion of panel C depicts the blot from the top of panel C reprobed with anti--actin after stripping. Panel D depicts lanes 5–8 from the blot in panel C stained with Ponceau S as an additional loading control. The amount of membrane protein loadedisindicatedbeloweachlane.Thecelltypestestedandcelltreatmentsareindicatedatthetop.HEKhFPR1,FPR1-transfectedHEK293cells.Westernblots in A, B, and C are from separate experiments. For A and B, the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific) was used, and for C the chemiluminescent Amersham Biosciences ECL Plus detection reagent (GE Healthcare) was used.

Article Snippet: PE-labeled mouse anti-FPR1 mAb (25 g/ml stock solution, catalog no. FAB3744P, clone 350418) and PElabeled IgG2A control (10 g/ml, catalog no. IC003P, clone 20102) were from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Stripping Membranes, Staining, Control, Membrane

Journal: Journal of Biological Chemistry

Article Title: The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

doi: 10.1074/jbc.m112.411181

Figure Lengend Snippet: FIGURE 3. FPR1 is resistant to ligand-induced internalization in lens epithelial cells. Cells were incubated for 30 min at 37 °C in the presence (dashed line) or absence (solid line) of 1 M fMLF and then stained with anti-FPR1. Unstimulated control cells were also stained with isotype control mAb (dotted line). A and B represent individual Fl-2 histograms showing the results of one representative experiment for the cell types indicated at the top. C, summary data from 2–5 independent experiments (1–2 samples for each individual condition). The number of experiments for each cell type is given above the bars.

Article Snippet: PE-labeled mouse anti-FPR1 mAb (25 g/ml stock solution, catalog no. FAB3744P, clone 350418) and PElabeled IgG2A control (10 g/ml, catalog no. IC003P, clone 20102) were from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

doi: 10.1074/jbc.m112.411181

Figure Lengend Snippet: FIGURE 4. FPR1 adopts distinct states in lens epithelial cells and FPR1-transfected HEK 293 cells; evidence from flow cytometry. A and B, staining of HEK 293-FPR1 cells (A) and FHL 124 cells (B) with anti-FPR1 is shown. Gray, anti-FPR1; black, isotype control. C and D, specific binding of fNLFNYK-Fl (10 nM) to HEK 293-FPR1 cells (C) and FHL 124 lens epithelial cells (D) is shown. Gray, total binding; black, nonspecific binding. Specific binding was calculated by subtraction of nonspecific binding (in the presence of 10 M fMLF) from total binding (10 nM fNLFNYK-Fl without fMLF). represents the difference between anti-FPR1 staining and isotype control background (A and B) or between total and nonspecific binding (C and D). The ratio of (HEK 293-FPR1)/ (FHL 124) is shown on the right. The data are from one representative experiment that was reproduced twice.

Article Snippet: PE-labeled mouse anti-FPR1 mAb (25 g/ml stock solution, catalog no. FAB3744P, clone 350418) and PElabeled IgG2A control (10 g/ml, catalog no. IC003P, clone 20102) were from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Flow Cytometry, Staining, Control, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

doi: 10.1074/jbc.m112.411181

Figure Lengend Snippet: FIGURE5.FPR1adoptsdistinctstatesinlensepithelialcellsandFPR1-transfectedHEK293cells;evidencefromconfocalmicroscopy.Cellswerestained with either antibody (anti-FPR1 or isotype control) or with 10 nM fNLFNYK-Fl (with or without excess unlabeled ligand) as indicated at the top of each column. The cell types are indicated on the left side of each row. Data are from representative experiments that were reproduced once.

Article Snippet: PE-labeled mouse anti-FPR1 mAb (25 g/ml stock solution, catalog no. FAB3744P, clone 350418) and PElabeled IgG2A control (10 g/ml, catalog no. IC003P, clone 20102) were from R&D Systems (Minneapolis, MN).

Techniques: Control

Journal: Journal of Biological Chemistry

Article Title: The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

doi: 10.1074/jbc.m112.411181

Figure Lengend Snippet: FIGURE 6. Characterization of high affinity binding sites for fluorescent FPR1 ligand fNLFNYK-Fl on lens epithelial cells. Specific binding (Œ) was calculated by subtraction of nonspecific binding (f, presence of peptide W at a 1000-fold excess compared with fNLFNYK-Fl) from total binding (only fNLFNYK-Fl, ●). A and B, total, nonspecific, and specific binding of fNLFNYK-Fl to HEK 293-FPR1 cells (A) and human neutrophils (B) is shown. C, comparison ofspecificbindingoffNLFNYK-FltoFHL124cellsanduntransfectedHEK293cellsisshown.D–F,saturationbindingoffNLFNYK-FltoFHL124lensepithelialcells showsthecompletecurvesfortotal,nonspecific,andspecificbinding(D)aswellasthecompletespecificbindingcurvealone(E)andthefirstpartofthespecific binding curve between 0 and 12 nM (F). All fluorescence data were recorded in the FL-1 PMT of a FACSCaliburTM flow cytometer at the same Fl-1 PMT settings (510 V). A–C show representative experiments with 2–3 replicates; the curves shown in D–F are the average of three independent experiments with 2–3 replicates.

Article Snippet: PE-labeled mouse anti-FPR1 mAb (25 g/ml stock solution, catalog no. FAB3744P, clone 350418) and PElabeled IgG2A control (10 g/ml, catalog no. IC003P, clone 20102) were from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Comparison, Fluorescence, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

doi: 10.1074/jbc.m112.411181

Figure Lengend Snippet: FIGURE 7. fNLFNYK-Fl binding sites are pharmacologically similar on lens epithelial cells, neutrophils, and FPR1-transfected HEK 293 cells. A–C, competition for binding of fNLFNYK-Fl (3 nM) by increasing concentrations of the FPR1 ligands fMLF (f), fNLFNYK (), or peptide W (●). MIP-1 (CCL3; MIP, macrophage inflammatory protein) () served as a non-binding negative control. Competition curves were recorded for HEK 293-FPR1 cells (A), FHL 124 lens epithelial cells (B), and human neutrophils (C). The curves contain the data of two independent experiments with duplicates. In each experiment all four ligands were tested at all three cell types on the same day under the same conditions.

Article Snippet: PE-labeled mouse anti-FPR1 mAb (25 g/ml stock solution, catalog no. FAB3744P, clone 350418) and PElabeled IgG2A control (10 g/ml, catalog no. IC003P, clone 20102) were from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Transfection, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

doi: 10.1074/jbc.m112.411181

Figure Lengend Snippet: FIGURE 8. FPR1 agonists induce Ca2 flux responses in lens epithelial cells in a pertussis toxin-sensitive manner. A–D, the cell types indicated at the top of each panel were incubated without (A and C) or with PTX (B and D), stimulated with substances listed in the insets, and analyzed by flow cytometry. ATP (100 M) was used as a positive control. Arrows identify the time when ligand was added. Each trace represents the average of 2–5 calcium flux responses. The effect of PTX on FPR1 agonist-mediated calcium signaling was confirmed by three additional independent plate reader experiments. E, fMLF potency and efficacy in FHL 124 cells (plate reader based experiment) is shown. The boxed inset in E shows the corresponding concentration-peak effect relationship generated from the kinetic curves with the resulting EC50 value. F, chemokines do not induce calcium flux in FHL 124 cells. RFU, relative fluorescence units.

Article Snippet: PE-labeled mouse anti-FPR1 mAb (25 g/ml stock solution, catalog no. FAB3744P, clone 350418) and PElabeled IgG2A control (10 g/ml, catalog no. IC003P, clone 20102) were from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Flow Cytometry, Positive Control, Concentration Assay, Generated, Fluorescence