anti pgp9 5  (Neuromics)

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: PTH treatment alters innervation in the degenerated spine. a Representative images of PGP9.5-positive fibers (upper row, Green) and CGRP-positive fibers (lower row, Red) in the lumbar vertebral body and endplate of aged mice treated with PTH or Veh for 1 or 2 months. Scale bar: 100 µm. Quantitative analysis of the length of PGP9.5-positive fibers ( b ) or CGRP-positive fibers ( c ) in the lumbar vertebral body of aged mice treated with PTH or Veh for 1 month or 2 months ( n ≥ 5, t -test). Representative images ( d ) and quantitative mean immunofluorescent (IF) intensity ( e ) of CGRP-positive neurons in the dorsal root ganglia (DRG) (L1-L2) of aged mice treated with PTH or Veh for 1 month or 2 months. Scale bar: 100 µm. ( n ≥ 6, t -test). f Protein levels of CGRP in DRG tissue (L1–L2) relative to the expression of GAPDH in aged mice treated with PTH or Veh for 1 or 2 months, respectively. ( n = 5). Representative images and quantitative analysis of the length of PGP9.5-positive ( g , h , Green) and CGRP-positive ( g , i , Red) fibers in the vertebral body and endplate of WT young mice 2 months after LSI surgery and treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 6, t -test). Representative images ( j ) and quantitative mean IF intensity ( k ) of CGRP-positive neurons in the DRG of WT young mice 2 months after LSI surgery and treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 5, t -test). DAPI stains nuclei blue. EP endplate, VB vertebral body. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Expressing

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Loss of PPR in osteoblasts ameliorates PTH response in endplate degeneration and pain behaviors during spine degeneration. BV/TV percentage ( a ), total porosity percentage ( b ), and total pore space ( c ) in PPR OCN −/− LSI mice with PTH or Veh treatment for 2 months. ( n = 5, t -test). Behavior evaluation of PPR OCN −/− LSI mice treated with PTH or Veh for 2 months included pressure tolerance of the lumbar spine region ( d , n ≥ 3, t -test), paw withdraw times of the hind paw response to 0.4 g Von Frey filament stimulation (10 times in total) ( e , n = 8, t -test), and latency of paw withdrawal post-thermal stimulation ( f , n ≥ 7, t -test). Representative images of PGP9.5 and CGRP-positive fibers ( g ), quantitative analysis of fiber length in the lumbar vertebral body of PPR OCN −/− LSI mice with PTH or Veh treatment ( h , i ). Scale bar: 100 µm. ( n ≥ 5, t -test). Representative images showing CGRP-positive neuron ( j ) and quantitative analysis of mean intensity of immunofluorescence ( k ) in the DRG of PPR OCN −/− LSI mice. Scale bar: 100 µm. ( n = 4, t -test). * P < 0.05, ** P < 0.01

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Immunofluorescence

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Osteoblasts respond to PTH treatment with increased Slit3 transcription and translation. mRNA expression of the Slit3 in the endplate tissue of WT young mice treated with Veh and aged mice treated with Veh or PTH for 1 month ( a ), and WT LSI mice treated with veh or PTH for 2 months ( b ) ( n = 3, one-way ANOVA with Tukey’s multiple comparisons test). c mRNA expression of the Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and subsequently treated with Veh or PTH at different doses for 3 days ( n = 3, t -test). d , e Protein expression level of Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and treated with Veh (PBS) or PTH for 3 days. ( n = 3, t -test). Representative images ( f ) and quantification ( g ) of IF staining of PGP9.5 positive primary DRG neuron fibers crossing the microgroove barrier (microfluid assay) cultured in condition medium (CM), CM + PTH, CM + PTH plus mouse Slit3 antibody (1 µg/mL), or CM + Veh (1X PBS) plus human recombinant Slit3 (1.25 µg/mL) for 1 week. Representative images showing co-immunostaining for OCN (green) and Slit3 (red) in the lumbar spine sections and quantitative analysis of number of Slit3 + cells in trabecular bone (TB) and endplate (EP) of aged mice ( h – j ) and PPR OCN −/− LSI mice ( k – m ) treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 5, t -test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Expressing, Cell Culture, Staining, Recombinant, Immunostaining

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Efficacy of PTH treatment is diminished in osteoblastic Slit3 knockout mice with spinal degeneration. Vertebral endplate bone structure analyses by micro-CT: bone volume per tissue volume (BV/TV) percentage ( a ), total porosity percentage ( b ), and total pore space ( c ) in Slit3 OCN −/− LSI mice with PTH (40 µg/kg/d) or Veh treatment for 2 months ( n = 7, t -test). Behavior evaluations included pressure tolerance of the lumbar spine assessed via the force threshold ( d ), total distance covered in spontaneous activity in 2 days ( e ), and latency of paw withdrawal post-thermal stimulation ( f ) for Slit3 OCN −/− LSI mice with PTH or Veh treatment for 2 months ( n = 7, t -test). g Western blot analysis of protein expression levels of β3 tubulin, CGRP, PGP9.5 relative to GAPDH in endplate tissue (L3-L5) in Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months. ( n = 5). Representative images of PGP9.5 (red) and CGRP (green)-positive fibers ( h ) and quantitative analysis of fiber length in the lumbar vertebral body of Slit3 OCN −/− LSI mice with PTH or Veh treatment ( i , j ) ( n = 5, t -tset). Scale bar: 100 µm. k Western blot analysis of protein expression levels of CGRP in DRG tissue (L1–L2), normalized to GAPDH expression, in Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months ( n = 5). Representative IF images showing CGRP-positive (green) neurons in DRG sections ( l ), followed by quantitative analysis of the mean IF intensity for CGRP ( m ) in the DRG of Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months ( n = 5, t -tset). Scale bar: 100 µm

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Knock-Out, Micro-CT, Activity Assay, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Clusterin protects against HFpEF by inhibiting UCHL1-mediated NLRP3 deubiquitylation and inflammasome activation

doi: 10.3389/fphar.2025.1704023

Figure Lengend Snippet: CLU interacted with UCHL1 and promoted NLRP3 degradation. (A) Western blot analysis of the interaction between CLU and UCHL1 detected by co-immunoprecipitation (Co-IP). (B) Western blot analysis of CLU, LC3B, p62, UCHL1, and NLRP3 in cells with or without CLU overexpression. β-actin was used as a loading control. Quantification of protein expression for (C) NLRP3 and (D) UCHL1. (E) The ubiquitination of NLRP3 promoted by CLU. (F) The ubiquitination of NLRP3 inhibited by UCHL1. (G) Western blot analysis of NLRP3 degradation after cycloheximide (CHX) treatment in CLU overexpressing cells. (H) Quantitative analysis of NLRP3 degradation.

Article Snippet: Protein samples were incubated with anti-CLU (Cell Signaling Technology, Cat# 34642), anti-NLRP3 (Proteintech, Cat# 68102-1-Ig), anti-Col1a2 (Abcam, Cat# ab308455), anti-UCHL1 (Proteintech, Cat# 14730-1-AP), anti-p62 (Proteintech, Cat# 18420-1-AP), anti-LC3B (Proteintech, Cat# 14600-1-AP).

Techniques: Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Control, Expressing, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: CGRP Enhances the Regeneration of Bone Defects by Regulating Bone Marrow Mesenchymal Stem Cells Through Promoting ANGPTL4 Secretion by Bone Blood Vessels

doi: 10.1002/advs.202522295

Figure Lengend Snippet: During bone defect repair, Cgrp − / − mice had lower bone angiogenesis than WT mice and exhibited delayed bone regeneration. (A) Representative micro‐CT images of the trabecular microstructure of WT mice before and after bone defect modeling. (B and C) Representative images and quantification of PGP9.5 (green)‐ and CGRP (red)‐stained femora from WT and Cgrp−/− mice after bone defect introduction. Scale bar, 100 µm. (D and E) Representative images and quantification in PGP9.5 (green)‐ and CD31 (red)‐stained femora from WT and Cgrp−/− mice after bone defects. Scale bar, 100 µm. (F and G) Representative images and quantification of CD31 (green)‐ and Emcn (red)‐stained femora from WT and Cgrp−/− mice after bone defect introduction. Scale bar, 100 µm. n = 3 mice in each group. (H–L) Representative micro‐CT images and quantitative micro‐CT analysis of the trabecular bone microarchitecture of WT and Cgrp−/− mice after bone defect introduction. Scale bar, 1 mm. (M) Representative images of hematoxylin–eosin staining in the bone generation area of WT and Cgrp−/− mice. Scale bar, 200 µm. (N and O) Representative images and quantitation of Runx2 (green) immunostaining in the bone generation area of WT and CGRP−/− mice. Scale bar, 100 µm. (P‐Q) Representative images of TRAP staining and quantification of TRAP‐positive cells on the trabecular bone surfaces of WT and CGRP−/− mice. Scale bar, 100 µm. n = 3 mice in each group. The data are shown as the mean ± standard deviation. * p < 0.05; ** p < 0.01; and *** p < 0.001 by Student's t ‐test. BV/TV, trabecular bone volume per tissue volume; Tb. N, trabecular number; Tb. Sp, trabecular separation; Tb. Th, trabecular thickness.

Article Snippet: Staining was carried out using primary antibodies against CD31 (Abcam, ab182981, 1:500), EMCN (Abcam, ab106100, 1:200), VEGFA (Boster Bio, BA0407, 1:200), Runx2 (Abcam, ab192256, 1:500), CGRP (Cell Signaling Technology, #14 959, 1:800), PGP9.5 (Proteintech, 14730‐1‐AP, 1:800), LEPR (Proteintech, 20966‐1‐AP, 1:50), and ANGPTL4 (Proteintech, 18374‐1‐AP, 1:500).

Techniques: Micro-CT, Staining, Quantitation Assay, Immunostaining, Standard Deviation