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: Chembiochem

Article Title: Design and Characterization of Prodrugged Anti‐CTLA‐4 Antibodies

doi: 10.1002/cbic.202500304

Figure Lengend Snippet: Structures of the masking reagent consist of: 1) a maleimide for conjugation, 2) a matriptase‐cleavable peptide (highlighted in purple), and 3) PEG to create steric hindrance, preventing the antibody from binding to the target.

Article Snippet: Prodrugged antibody was incubated with human matriptase (30:1 molar ratio, R&D system, 3946‐SE‐010) in 100 mM Tris buffer, pH7.6 at 37C.

Techniques: Conjugation Assay, Binding Assay