Evaluation of antibodies directed against human protease-activated receptor-2.

Summary of "Evaluation of antibodies directed against human protease-activated receptor-2."

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by intramolecular docking of a tethered ligand that is released by the actions of proteases, mainly of the serine protease family. Here, we evaluate four commercially available anti-PAR2 antibodies, SAM11, C17, N19 and H99, demonstrating marked differences in the ability of these reagents to detect the target receptor in Western blot, immunocytochemical and flow cytometry applications. In Western blot analysis, we evaluated antibody reactivity against both ectopic and endogenous receptors. Against material from transfected cells, we show that SAM11 and N19, and to a lesser extent C17, but not H99, are able to detect ectopic PAR2. Interestingly, these Western blot analyses indicate that N19 and C17 detect conformations of ectopic PAR2 distinct to those recognised by SAM11. Significantly, our data also indicate that Western blot signal detected by SAM11 and C17, and much of the signal detected by N19, against cells endogenously expressing PAR2 is non-specific. Despite confounding non-specific signals, we were able to discern N19 reactivity against endogenous PAR2 as a broad smear that we also observed in ectopically expressing human and mouse cells and that is sensitive to loss of N-glycosylation. In immunocytochemistry analysis, each antibody is able to detect ectopic PAR2 although it appears that H99 detects only a subset of the ectopically expressed receptor. In addition, SAM11 and N19 are able to detect both ectopic and endogenous cell surface PAR2 by flow cytometry. In summary: (1) each antibody can detect ectopic PAR2 by immunocytochemical analysis with SAM11 and N19 suitable for cell surface detection of both ectopic and endogenous receptor by flow cytometry; (2) in Western blot analysis, N19, SAM11 and C17 can detect ectopically expressed PAR2, with only N19 able to detect the endogenous receptor by this technique and (3) in each of these approaches, appropriate controls are essential to ensure that non-specific reactivity is identified.


Mater Medical Research Institute, Aubigny Place, Raymond Terrace, South Brisbane, QLD, 4101, Australia.

Journal Details

This article was published in the following journal.

Name: Naunyn-Schmiedeberg's archives of pharmacology
ISSN: 1432-1912


DeepDyve research library

PubMed Articles [29253 Associated PubMed Articles listed on BioPortfolio]

Protease-activated receptor-2 suppresses interleukin (IL)-10 expression in B cells via up regulating Bcl2L12 in patients with allergic rhinitis.

The function of interleukin (IL)-10 producing B cells (B10 cell) is compromised in patients with allergic diseases. Protease-activated receptor (PAR)-2 has immune regulatory functions. This study aims...

Establishment of a SD rat model of vulvar lichen simplex chronicus and detection of the expression of protease activated receptor 2.

To establish a SD rat model of vulvar lichen simplex chronicus (LSC) and investigate the expression of protease activated receptor 2 (PAR2) in the genital skin.

Protease-activated Receptor 1 Plays a Proinflammatory Role in Colitis by Promoting Th17-related Immunity.

Proteolytic cleavage of protease-activated receptor 1 (PAR1) can result in potent downstream regulatory effects on inflammation. Although PAR1 is expressed throughout the gastrointestinal tract and ac...

Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies.

Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1-7) protective axis of renin-angiotensin system. Because the r...

Role of Protease-Activated Receptor 2 in Regulating Focal Segmental Glomerulosclerosis.

Background /Aims: The underlying mechanisms leading to focal segmental glomerulosclerosis (FSGS) are lacking. In this report, we examined the role of protease-activated receptors (PARs) subtype PAR2 a...

Clinical Trials [7020 Associated Clinical Trials listed on BioPortfolio]

Protease Activated Receptor-2 and Gastrointestinal Dysfunction in Critical Illness

Gastrointestinal (GI) dysfunction affects up to 50% of medical and surgical critically ill children. GI dysfunction, specifically gastric dysmotility and loss of epithelial barrier integri...

Nelfinavir in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease in which the body's immune system attacks different parts of the body. SLE is characterized by inflammation that leads t...

Trial of Donor Lymphocyte Infusion (DLI) and Activated DLI Following Relapse After Allogeneic Stem Cell Transplant

This study is for patients with relapsed of disease after allogeneic bone marrow The donor's T cells are activated by exposure to 2 compounds or antibodies that bind (or stick to) two com...

Evaluation of Recombinant Factor VIIa in Patients With Severe Bleeding

This trial is conducted globally. The purpose of the trial is to evaluate that activated recombinant human factor VII is safe and effective in severely injured trauma patients by assessing...

Activated T-cell Therapy, Low-Dose Aldesleukin, and Sargramostim in Treating Patients With Ovarian, Fallopian Tube, or Primary Peritoneal Cancer That is Stage III-IV, Refractory, or Recurrent

This phase I trial studies the side effects and best dose of activated T-cell therapy when given together with low-dose aldesleukin and sargramostim in treating patients with ovarian, fall...

Medical and Biotech [MESH] Definitions

A 77-kDa subcomponent of complement C1, encoded by gene C1S, is a SERINE PROTEASE existing as a proenzyme (homodimer) in the intact complement C1 complex. Upon the binding of COMPLEMENT C1Q to antibodies, the activated COMPLEMENT C1R cleaves C1s into two chains, A (heavy) and B (light, the serine protease), linked by disulfide bonds yielding the active C1s. The activated C1s, in turn, cleaves COMPLEMENT C2 and COMPLEMENT C4 to form C4b2a (CLASSICAL C3 CONVERTASE).

A 80-kDa subcomponent of complement C1, existing as a SERINE PROTEASE proenzyme in the intact complement C1 complex. When COMPLEMENT C1Q is bound to antibodies, the changed tertiary structure causes autolytic activation of complement C1r which is cleaved into two chains, A (heavy) and B (light, the serine protease), connected by disulfide bonds. The activated C1r serine protease, in turn, activates COMPLEMENT C1S proenzyme by cleaving the Arg426-Ile427 bond. No fragment is released when either C1r or C1s is cleaved.

A G-protein-coupled, proteinase-activated receptor that is expressed in a variety of tissues including ENDOTHELIUM; LEUKOCYTES; and the GASTROINTESTINAL TRACT. The receptor is activated by TRYPSIN, which cleaves off the N-terminal peptide from the receptor. The new N-terminal peptide is a cryptic ligand for the receptor. The uncleaved receptor can also be activated by the N-terminal peptide present on the activated THROMBIN RECEPTOR and by small synthetic peptides that contain the unmasked N-terminal sequence.

Enzyme of the human immunodeficiency virus that is required for post-translational cleavage of gag and gag-pol precursor polyproteins into functional products needed for viral assembly. HIV protease is an aspartic protease encoded by the amino terminus of the pol gene.

Antibodies elicited in a different species from which the antigen originated. These antibodies are directed against a wide variety of interspecies-specific antigens, the best known of which are Forssman, Hanganutziu-Deicher (H-D), and Paul-Bunnell (P-B). Incidence of antibodies to these antigens--i.e., the phenomenon of heterophile antibody response--is useful in the serodiagnosis, pathogenesis, and prognosis of infection and latent infectious states as well as in cancer classification.

Quick Search

DeepDyve research library

Searches Linking to this Article