nmol l tumor necrosis factor  (R&D Systems)

Journal: IBRO Neuroscience Reports

Article Title: TGF-β1 modulates PFC glial cell activation to facilitate exercise-induced analgesia in mice with spared nerve injury

doi: 10.1016/j.ibneur.2026.03.009

Figure Lengend Snippet: Exercise modulates TGF-β1 expression in the prefrontal cortex (PFC) of mice 24 days after spared nerve injury (SNI). (a) Representative Western blot images of TGF-β receptor I (TGF-βR1) and TGF-β1 in the PFC. Tissue lysates from all experimental groups (SHAM, SHAME, SNI, SNIE) and recombinant human TGF-β1 (non-reduced and reduced) were loaded on the same SDS–PAGE gel, transferred to a single membrane, and probed with the same TGF-β1 antibody under identical exposure conditions. The recombinant protein (250 ng per lane) served as a positive control to verify the molecular weights of the dimeric (25 kDa) and monomeric (12.5 kDa) forms of TGF-β1. GAPDH was used as the loading control. (b-d) Quantitative Western blot analyses of (b) TGF-βR1, (c) dimeric TGF-β1 (25 kDa), and (d) monomeric TGF-β1 (12.5 kDa) expression levels in tissue lysates. Data are presented as mean ± SEM (n = 3). ** P < 0.01 vs. SHAM group; ## P < 0.01 vs. SNI group.

Article Snippet: To validate the specificity of the TGF-β1 antibody, Recombinant human TGF-β1 protein (Catalog # 240-B, R&D Systems, USA) was used as a positive control.

Techniques: Expressing, Western Blot, Recombinant, SDS Page, Membrane, Positive Control, Control

Journal: IBRO Neuroscience Reports

Article Title: TGF-β1 modulates PFC glial cell activation to facilitate exercise-induced analgesia in mice with spared nerve injury

doi: 10.1016/j.ibneur.2026.03.009

Figure Lengend Snippet: At 24 d after SNI, mouse PFC astrocytes were activated and microglia were unchanged. (a)Western blotting analysis of changes in GFAP and Iba1 expression in PFC (n = 3); (b) Quantification of GFAP in PFC; (c) Quantification of Iba1 in PFC; (d) MFI representative images of GFAP in PFC; (e) MFI representative image of Iba1 in PFC; (f) Quantification of GFAP in PFC. Values represent mean ± SEM (Scale bar =75μm, 9 PFC sections from 3 mice per group); (g) Quantification of Iba1 in PFC. Values represent mean ±SEM (Scale bar = 75μm, 9 PFC sections from 3 mice per group). Values represent the mean ±SEM. * P < 0.05, ** P < 0.01, compared with SHAM group; # P < 0.05, ## P < 0.01, compared with SNI group, the difference was statistically significant; (h) Representative MFI images of changes in the colocalization of TGF-β1(red) and astrocytes (green) in the PFC; (i) Quantification of TGF-β1 and astrocytes in PFC. Values represent the mean ± SEM (Scale bar =100μm, nine PFC sections from three mice per group). * P < 0.05 versus the SHAM group; # P < 0.05 versus the SNI group.

Article Snippet: To validate the specificity of the TGF-β1 antibody, Recombinant human TGF-β1 protein (Catalog # 240-B, R&D Systems, USA) was used as a positive control.

Techniques: Western Blot, Expressing

Journal: IBRO Neuroscience Reports

Article Title: TGF-β1 modulates PFC glial cell activation to facilitate exercise-induced analgesia in mice with spared nerve injury

doi: 10.1016/j.ibneur.2026.03.009

Figure Lengend Snippet: TGF-βRI inhibition reverses exercise-induced analgesia and modulates glial activation in the PFC. (a, b) Time course of mechanical and cold hyperalgesia tests (n = 9). The green shading indicates the duration of the exercise intervention, and the green vertical lines denote the timing of intrathecal injections. Data are presented as mean ± SEM. ** P < 0.01 versus the SNIE group, # P < 0.05, ## P < 0.01 vs. SC group. (c) Representative Western blot images of TGF-βR1 and TGF-β1 in the PFC. Tissue lysates from SC and SA groups and recombinant human TGF-β1 (100 ng per lane) (non-reduced and reduced) were loaded on the same SDS–PAGE gel, transferred to a single membrane, and probed with the same TGF-β1 antibody in a single exposure without splicing. The recombinant protein served as a positive control to verify the molecular weights of the dimeric (25 kDa) and monomeric (12.5 kDa) forms of TGF-β1. GAPDH was used as the loading control. (d-f) Quantitative analysis of (d) TGF-βR1, (e) dimeric TGF-β1 (25 kDa), and (f) monomeric TGF-β1 (12.5 kDa) expression levels (n = 3). (g-i) Western blot analysis of glial markers. (g) Representative images of GFAP and Iba1 with GAPDH control. Quantitative analysis of (h) GFAP and (i) Iba1 expression levels (n = 3). (j, k) Representative immunofluorescence images showing the expression of (j) GFAP and (k) Iba1 in the PFC. Scale bar = 75 μm. (l, m) Quantification of the mean fluorescence intensity (MFI) for (l) GFAP and (m) Iba1 (n = 9 sections from 3 mice per group). Data in bar graphs are presented as mean ± SEM. * P < 0.05, ** P < 0.01 vs. SC group. SC: Spared nerve injury with exercise training followed by intrathecal (i.t.) injection of saline; SA: Spared nerve injury with exercise training followed by i.t. injection of the TGF-βRI inhibitor.

Article Snippet: To validate the specificity of the TGF-β1 antibody, Recombinant human TGF-β1 protein (Catalog # 240-B, R&D Systems, USA) was used as a positive control.

Techniques: Inhibition, Activation Assay, Western Blot, Recombinant, SDS Page, Membrane, Positive Control, Control, Expressing, Immunofluorescence, Fluorescence, Injection, Saline

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