rabbit polyclonal anti par1 antibody  (Alomone Labs)

Journal: Allergy, Asthma, and Clinical Immunology : Official Journal of the Canadian Society of Allergy and Clinical Immunology

Article Title: A novel pathogenetic factor of laryngeal attack in hereditary angioedema? Involvement of protease activated receptor 1

doi: 10.1186/s13223-022-00699-7

Figure Lengend Snippet: Parameters of primary antibodies used in the study

Article Snippet: PAR1 Rabbit anti-Human Polyclonal (N-Terminus) Antibody, 1:1000 , LifeSpan BioSciences , LS-A2583-50.

Techniques:

Journal: Allergy, Asthma, and Clinical Immunology : Official Journal of the Canadian Society of Allergy and Clinical Immunology

Article Title: A novel pathogenetic factor of laryngeal attack in hereditary angioedema? Involvement of protease activated receptor 1

doi: 10.1186/s13223-022-00699-7

Figure Lengend Snippet: Expression of PAR1 in HAE patient and control. Samples were incubated with rabbit anti-PAR1 antibody followed by peroxidase conjugated goat anti-rabbit secondary antibody. The immune reaction was developed by DAB and counterstained with hematoxylin. HAE patient’s ( A , C , E ) and control’s ( B , D , F ) epiglottis ( A , B ), true vocal cord ( C , D ) and false vocal cord ( E , F ) regions are shown. Black arrows indicate endothelial cells and white arrows indicate epithelial cells. Scale bar: 50 μm. Expression pattern of PAR1 in different anatomical locations is compared as staining intensity scores. Green bars represent the control subject’s values, red bars represent the HAE patient’s values. Sum scores ( I ) were calculated by the summation of epithelial cells ( G ), endothelial cells ( H ), leukocytes and interstitial space staining intensity scores (individually ranged from 0 to 3). (EPI: epiglottis, TVC: true vocal cord, FVC: false vocal cord.)

Article Snippet: PAR1 Rabbit anti-Human Polyclonal (N-Terminus) Antibody, 1:1000 , LifeSpan BioSciences , LS-A2583-50.

Techniques: Expressing, Incubation, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: C4a is a putative agonist for protease-activated receptors (PAR)1 and PAR4. Screening of the gpcrMAX panel in agonist mode reveals that PAR1 and PAR4 meet the selective criteria as putative targets for C4a. Dash line represents 30% of activation. The data represent the mean ± SD of duplicate samples.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: C4a acts as an agonist for PAR1 and PAR4. CHO-K1 cells expressing either PAR1 or PAR4 were seeded in a 96-well plate and stimulated with PAR1 agonist TFLLR-NH2, PAR4 agonist AY-NH2, human C4a, C3a, or C5a. After stimulation, the chemiluminescent signal was detected. Culture medium alone was used as a control. (A) C4a and PAR1 agonist TFLLR-NH2 dose-dependently activate PAR1. (B) PAR1 antagonist RWJ56110 dose-dependently inhibits C4a-induced PAR1 activation. (C) C4a and PAR4 agonist AY-NH2 dose-dependently activate PAR4. (D) PAR4 antagonist tcY-NH2 has no effect on C4a-induced PAR4 activation. The data are expressed as fold change compared with control or relative light unit (RLU) and represent the mean ± SE of three independent experiments.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: The N-terminal area of C4a is essentially involved in the activation of PAR1 and PAR4. In contrast to the anaphylatoxins (C3a and C5a) and their corresponding receptors, the desarginated form of C4a (i.e., C4a-desArg) remains to activate both PAR1 (A) and PAR4 (B). Moreover, the C-terminal 20 amino acids of C4a (i.e., C4a-CT20) do not exert agonistic activity on either PAR1 or PAR4. The data are expressed as fold change compared with the control group and represent the mean ± SE of three independent experiments.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Activity Assay, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: Expression and purification of human His6-C4a for colocalization studies on PAR1/4-expressing cells. (A) Western blots reveal the expression of PAR1 in CHO-K1 PAR1 cells. (B) Western blots reveal the expression of PAR4 in CHO-K1 PAR4 cells. The cell lysate of CHO-K1 cells was used as control. (C) Recombinant human C4a fused to an S-tag and a His6-tag was expressed in E. coli strain Rosetta-gami B (DE3) Lys-S and purified using a His-Trap column and S-protein agarose (EMD). His6-S-tag-C4a, S-tag-C4a, and C4a were analyzed by SDS/PAGE (15%) and stained with Coomassie blue (white spacers indicate noncontiguous lanes from different Coomassie blue staining gels). (D) Amino acid sequence of the recombinant human 6His-C4a fusion protein used for the colocalization experiments. The protein containing 6His and S-tags was expressed in E. coli using pET-32a-hC4a construct based on a previous publication (9). Functional parts of the protein are marked as follows: blue, 6His-tag; red, enterokinase cleavage site; yellow, S-tag; and green, human C4a.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Expressing, Purification, Western Blot, Control, Recombinant, SDS Page, Staining, Sequencing, Construct, Functional Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: On CHO-K1 cells expressing either PAR1 or PAR4, recombinant human C4a colocalizes with the corresponding receptor. His6-C4a (red, anti-6His Ab) colocalizes with PAR1 (A; green, anti-PAR1 Ab) or PAR4 (B; green, anti-PAR4 Ab) on CHO-K1 cells expressing PAR1 or PAR4, respectively, but does not bind to wild-type CHO-K1 cells (Fig. S4A). The experiment was performed three times with similar results, and one representative experiment is shown.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Expressing, Recombinant

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: Colocalization of human His6C4a with PAR1 and PAR4 in CHO-K1 and HMEC-1 cells. (A) No colocalization of 6His-C4a with PAR1 (Upper) or PAR4 (Lower) was observed in wild-type CHO-K1 cells (for experiments using PAR1- and PAR4-expressing CHO-K1 cells see Fig. 3). (B) His6-C4a colocalizes with PAR1 in HMEC-1 cells. (C) His6-C4a colocalizes with PAR4 in HMEC-1 cells. The experiment was performed three times with similar results, and one representative experiment is shown.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: In human HMEC-1 endothelial cells, C4a increases ERK phosphorylation through a Gαi-independent signaling pathway. (A) C4a dose-dependently enhances ERK phosphorylation. (B) PAR1 antagonist RWJ56110 (10 μM), but not pertussis toxin (PTX; 0.3 μg/mL, 10 h), inhibits ERK activation upon C4a exposure (7 min). The data are expressed as fold change in densitometry of Western blots from the control group and represent the mean ± SE of three independent experiments. (n = 3; *P < 0.05 vs. control; pairwise two-sided Student’s t test.)

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: ERK activation by C4a on human endothelial cells. (A) C4a time-dependently induces ERK activation in HMEC-1 cells. The data are expressed as −fold change compared with the control group and represent the mean ± SE of three independent experiments. (B) In EA.hy926 cells, C4a induces ERK activation, and the PAR1 antagonist RWJ56110 (10 μM), but not the PAR4 antagonist tcY-NH2 or pertussis toxin (0.3 μg/mL for 10 h), inhibits ERK activation after C4a treatment for 7 min. The experiment was performed three times with similar results, and one representative experiment is shown. (C) PAR4 agonist AY-NH2 and thrombin activate ERK on EA.hy926 cells. The experiment was performed three times with similar results, and one representative experiment is shown.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: PAR1 and PAR4 are involved in C4a-induced ERK activation in human endothelial cells. (A) Selective PAR4 receptor agonist peptide AY-NH2 (AYPGKF-NH2) significantly increases ERK phosphorylation in HMEC-1 cells. The data are expressed as fold change in densitometry of Western blots from the control group and represent the mean ± SE of three independent experiments. (n = 3; **P < 0.01 vs. control; pairwise two-sided Student’s t test.) (B) Pretreatment with PAR4 polyclonal blocking antibody (H-120) significantly decreases ERK phosphorylation by C4a treatment (7 min) in HMEC-1 cells. The experiment was performed three times. The data are expressed as fold change in densitometry of Western blots from the control group and represent the mean ± SE of three independent experiments. (n = 3; **P < 0.01 vs. control; pairwise two-sided Student’s t test.) White spacers indicate noncontiguous lanes of the same Western blot. (C) HMEC-1 cells were pretreated with the PAR1 antagonist RWJ56110 (10 mM), PAR4 blocking antibody (4 μg/mL), or a combination of both inhibitors. The data are expressed as fold change compared with the control group and represent the mean ± SE of three independent experiments. [n = 3; **P < 0.01 vs. C4a treatment (7 min); pairwise two-sided Student’s t test.]

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control, Blocking Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: C4a increases [Ca2+]i and endothelial permeability via PAR1 activation. (A) C4a dose-dependently increases [Ca2+]i in HMEC-1 cells. The data are expressed as relative fluorescence [Δ(ΔRFU)] and represent the mean ± SE of four to seven independent experiments. (B) PAR1 antagonist RWJ56110 (10 μM) significantly inhibits C4a-mediated elevation of [Ca2+]i. The data are expressed as relative fluorescence [Δ(ΔRFU)] and represent the mean ± SE of seven independent experiments. (n = 7; **P < 0.01 vs. control; pairwise two-sided Student’s t test.) (C) Phospholipase C inhibitor U73122 inhibits C4a-induced elevation of [Ca2+]i. The data are expressed as relative fluorescence [Δ(ΔRFU)] and represent the mean ± SE of eight independent experiments (n = 8; **P < 0.01 vs. control; pairwise two-sided Student’s t test). (D) In EA.hy926 cells, C4a dose-dependently increases endothelial permeability, and the PAR1 antagonist RWJ56110 significantly inhibits C4a-induced endothelial permeability. The data are expressed as OD at 650 nm and represent the mean ± SE of four independent experiments. [n = 4; *P < 0.05 vs. control; **P < 0.01 vs. control; ##P < 0.01 vs. C4a (300 nM); pairwise two-sided Student’s t test.] (E) C4a-induced stress fiber formation is significantly decreased by pretreatment with PAR1 antagonist RWJ56110. The experiment was performed three times with similar results, and one representative experiment is shown.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Permeability, Activation Assay, Fluorescence, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: C4a disrupts endothelial barrier function in HMEC-1 cells. (A) In HMEC-1 endothelial cells, C4a dose-dependently increases endothelial permeability, and the PAR1 antagonist RWJ56110 significantly inhibits C4a-induced endothelial permeability. The data are expressed as OD at 650 nm and represent the mean ± SE of three independent experiments. [n = 3; **P < 0.01 vs. control; ##P < 0.01 vs. C4a (300 nM); pairwise two-sided Student’s t test.] (B) C4a-induced stress fiber formation is significantly decreased by pretreatment with PAR1 antagonist RWJ56110. The experiment was performed three times with similar results, and one representative experiment is shown.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Permeability, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4

doi: 10.1073/pnas.1707364114

Figure Lengend Snippet: C4a-induced PAR1 and PAR4 activation signals are not caused by thrombin contamination. The thrombin activity assay reveals that the C4a preparation used in this study fails to activate the chromogenic thrombin substrate even at a concentration of 3 μM, whereas thrombin itself shows activity at less than 1 nM, which can be reversed by adding a thrombin inhibitor. The data are expressed as fold change compared with the control group and represent the mean ± SE of three independent experiments.

Article Snippet: Rabbit anti-human PAR1 (H-111) and PAR4 (H-120) polyclonal antibodies were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Activity Assay, Concentration Assay, Control