par2 activating matriptase  (R&D Systems)

Journal: Pain Reports

Article Title: Monoclonal antibody inhibition of PAR2 reduces phenotype severity and pain in murine inflammatory bowel disease

doi: 10.1097/PR9.0000000000001446

Figure Lengend Snippet: Colonic PAR2 activation by 2F elicits LSN responses that are mediated by endosomal internalization, PKA and PKC. (A) Experimental framework for LSN recordings showing a cannulated colon in a recording chamber with the LSN aspirated into a suction electrode. (B) Representative action potentials recorded from LSN afferent fiber prestimulation. (C and D) 100 µM 2F application elicits LSN responses illustrated by (C) action potential traces prestimulation and 15 minutes poststimulation and (D) change in LSN firing rate over time after stimulation via the luminal inflow (vertical dotted line). 2F-stimulation increased LSN firing (E and F). Timeline and peak change in firing rate after 100 µM 2F application (vertical dotted line) in tissue pretreated with (E) 50 µM PitStop2 (PS2) inhibitor for endosomal internalization or negative control PitNot2 (PN2) and (F) 100 µM H-89 dihydrochloride (H-89) and bisindolylmaleimide (GFX), PKA and PKC inhibitors or DMSO vehicle. (E) PS2 as well as separate and simultaneous pretreatment with H-89 and GFX reduced the peak response to 2F. (D and E) Independent samples t test comparing the peak change in firing rate (N = 5–9). (F) One-way ANOVA with post-hoc FDR-corrected independent samples t test (N = 5–6). * P < 0.05, ** P < 0.01, *** P < 0.001 **** P < 0.0001. Data are presented as mean ± SD. LSN, lumbar splanchnic nerve; PAR2, protease-activated receptor 2; PKA, protein kinase A; PKC, protein kinase C.

Article Snippet: When measuring the inhibitory concentration 50 (IC50), cells received PAR650097 mIgG or hIgG or isotype control antibodies, for 60 minutes at room temperature before addition of PAR2 activating matriptase (3946-SEB-010, R&D Systems, Abingdon, United Kingdom).

Techniques: Activation Assay, Negative Control

Journal: Pain Reports

Article Title: Monoclonal antibody inhibition of PAR2 reduces phenotype severity and pain in murine inflammatory bowel disease

doi: 10.1097/PR9.0000000000001446

Figure Lengend Snippet: PAR2 activation in the colon by 2F sensitizes LSN responses to mechanical and chemical stimulation of the colon via endosomal internalization, PKA and PKC. LSN action potential firing after mechanical distention: gradually increasing intraluminal pressure from 0 to 80 mm Hg, and chemical application: 1 mM cinnamaldehyde and 1 µM capsaicin are illustrated in (A). (B) Responses to distention were quantified through the change in firing rate over increasing discrete pressure values. (C and D) Responses to chemical stimuli were quantified through the change in firing rate over time after application (vertical dotted line). 100 µM 2F stimulation, but not vehicle control, elicited sensitization of the LSN to (B) distention, (C) cinnamaldehyde, and (D) capsaicin. Pretreatment with PS2 before 2F stimulation reduced subsequent LSN responses to distention, cinnamaldehyde, and capsaicin compared with PN2. Pretreatment with H-89 and GFX, applied either simultaneously or individually, also reduced subsequent peak LSN responses to distention, cinnamaldehyde, and capsaicin compared with DMSO vehicle. (B–D) Independent samples t tests and one-way ANOVA with post-hoc FDR-corrected independent samples t test (N = 5–9). * P < 0.05, ** P < 0.01, **** P < 0.0001. Data are presented as means ± SD. LSN, lumbar splanchnic nerve; PAR2, protease-activated receptor 2; PKA, protein kinase A; PKC, protein kinase C.

Article Snippet: When measuring the inhibitory concentration 50 (IC50), cells received PAR650097 mIgG or hIgG or isotype control antibodies, for 60 minutes at room temperature before addition of PAR2 activating matriptase (3946-SEB-010, R&D Systems, Abingdon, United Kingdom).

Techniques: Activation Assay, Control

Journal: Cellular and Molecular Neurobiology

Article Title: Procoagulant Extracellular Vesicles Increase Neuronal Tau expression, Metabolism and Processing Through Tissue Factor and Protease Activated Receptor 2

doi: 10.1007/s10571-025-01658-7

Figure Lengend Snippet: Examination of the mechanism of influence of TF on Tau protein expression and phosphorylation in differentiated SH-SY5Y cells. SH-SY5Y (2 × 10 5 ) were treated with recombinant relipidated Innovin TF (0.65 ng/ml) together with or without human fVIIa (5 nM) or fVIIa alone. In some experiments, the TF aliquots were pre-incubated for 1 h, with 10H10 antibody (20 µg/ml) capable of blocking TF signalling, HTF-1 antibody (20 µg/ml) to block TF-fVIIa protease/procoagulant activity, or a mouse control isotype IgG antibody (20 µg/ml; not shown). In other experiments, fVIIa was pre-incubated for 1 h with the chemical inhibitor PCI27483 (10 µg/ml). Alternatively, the neuronal cells were treated with AIIB2 antibody (20 µg/ml) to block β1-integrin signalling, SAM11 antibody (20 µg/ml) capable of blocking PAR2 signalling, or PAR2-activating peptide (PAR2-AP; 20 µM) to induce PAR2 signalling. Cells were harvested at 24 h, and cellular lysates (10 µg protein) were examined for Tau and phospho-Thr181 Tau by western blot analysis. All values were normalised against the respective GAPDH and for comparison, all ratios were calculated against the average from the non-treated cells ± the calculated standard deviation. The number of experiments is shown in each column, and all data groups were determined to have normal distributions and are shown in each column. A SH-SY5Y protein electrophoresis, B SH-SY5Y phospho-Thr181 Tau electrophoresis, C SH-SY5Y Tau protein ratio, and D SH-SY5Y phospho-Thr181 Tau ratio

Article Snippet: Alternatively, the neuronal cells were treated with a rat anti-human antibody (20 μg/ml; AIIB2; Merck KGaA) to block β1-integrin signalling, a mouse anti-human PAR2 antibody, SAM11 (20 μg/ml; Santa Cruz Biotechnology, Heidelberg, Germany), capable of blocking PAR2 signalling, or PAR2-activating peptide (PAR2-AP; 20 μM) to induce PAR2 signalling.

Techniques: Expressing, Phospho-proteomics, Recombinant, Incubation, Blocking Assay, Activity Assay, Control, Western Blot, Comparison, Standard Deviation, Protein Electrophoresis, Electrophoresis

Journal: Cellular and Molecular Neurobiology

Article Title: Procoagulant Extracellular Vesicles Increase Neuronal Tau expression, Metabolism and Processing Through Tissue Factor and Protease Activated Receptor 2

doi: 10.1007/s10571-025-01658-7

Figure Lengend Snippet: Time-course analysis of the Tau protein fragments in differentiated SH-SY5Y cells, following treatment with TF . SH-SY5Y (2 × 10 5 ) were treated with as single dose of recombinant relipidated Innovin TF (0.65 ng/ml) together with human fVIIa (5 nM). In some experiments, the TF aliquots were pre-incubated for 1 h, with 10H10 antibody (20 µg/ml) capable of blocking TF signalling, HTF-1 antibody (20 µg/ml) to block TF-fVIIa protease/procoagulant activity, or a mouse control isotype IgG antibody (20 µg/ml; not shown). Alternatively, the neuronal cells were treated with AIIB2 antibody (20 µg/ml) to block β1-integrin signalling, or SAM11 antibody (20 µg/ml) capable of blocking PAR2 signalling. Sets of cells were harvested at A 48 h and D at 72 h and cellular lysates (10 µg protein) were examined for Tau by western blot analysis. All values were normalised against the respective GAPDH (see Supplementary Fig. 6 A and B) and for comparison, all ratios were calculated against the average from the non-treated cells ± the calculated standard deviation. The data were obtained from 6 biological experiments, and all data groups were determined to have normal distributions. A Electrophoresis at 48 h, B calculated ratios of 50 kDa bands at 48 h, C calculated ratios of 30–35 kDa bands at 48 h, D electrophoresis at 72 h, E calculated ratios of 50 kDa bands at 72 h, F calculated ratios of 40 kDa bands at 72 h, G calculated ratios of 30–35 kDa bands at 72 h

Article Snippet: Alternatively, the neuronal cells were treated with a rat anti-human antibody (20 μg/ml; AIIB2; Merck KGaA) to block β1-integrin signalling, a mouse anti-human PAR2 antibody, SAM11 (20 μg/ml; Santa Cruz Biotechnology, Heidelberg, Germany), capable of blocking PAR2 signalling, or PAR2-activating peptide (PAR2-AP; 20 μM) to induce PAR2 signalling.

Techniques: Recombinant, Incubation, Blocking Assay, Activity Assay, Control, Western Blot, Comparison, Standard Deviation, Electrophoresis