mouse psmad2  (Cell Signaling Technology Inc)

Journal: Bone Research

Article Title: TGF-β-induced fibrotic scar formation limits recovery of spinal cord injury

doi: 10.1038/s41413-026-00507-7

Figure Lengend Snippet: Conditional knockout of Tgfβ1 in macrophage lineage cells reduces pSmad2 levels and promotes functional recovery in SCI mice. a – d ELISA analysis showing the concentration of TGF-β in the spinal cord and the serum after SCI in each mouse for the groups indicated (* P < 0.05, ** P < 0.01, n = 4). e Representative Western blots showing the activation of phosphorylated Smad (pSmad) signaling. f Representative images of immunofluorescent staining of pSmad2 + (red), platelet-derived growth factor receptor-β + (PDGFR-β + ) (green) pericytes , and DAPI (blue) staining of nuclei at 7 days after SCI. Scale bar, 10 µm. g Representative immunofluorescence images of PDGFR-β + (green) pericytes and DAPI (blue) staining of nuclei at 2 weeks after SCI. Scale bars, 200 µm. h Quantitative analysis of the number of pSmad2 + cells (**** P < 0.000 1, n = 6). i Quantitative analysis of the number of PDGFR-β + cells (** P < 0.01, n = 6). j , k Illustrations of the hot plate test and the von Frey test. l Quantitative analysis of hindpaw withdrawal time responding to temperature (hot plate test, ** P < 0.01, n = 8). m , n Quantitative analysis of hindpaw withdrawal frequency responding to mechanical stimulation (von Frey test, 0.7 mN and 3.9 mN, * P < 0.05, ** P < 0.01, n = 8). o Quantitative analysis of Basso Mouse Scale (BMS) score between Tgfb1 flox / flox control mice after SCI, Tgfb1 LysM-cre −/− mice after SCI, and Tgfb1 flox/flox control mice without SCI (* P < 0.05, n = 8). PWF paw withdrawal frequency. LF left forepaw, RF right forepaw, RH right hindpaw. Statistical significance was determined by multifactorial ANOVA, and all data are shown as means ± standard deviations. Source data are provided as a Source Data file

Article Snippet: We used antibodies recognizing mouse pSmad2 (1:1 000, Cell Signaling Technology, Inc., Danvers, MA, USA) and Smad2 (1:1 000, Cell Signaling Technology, Inc.) to examine the protein concentrations in the lysates.

Techniques: Knock-Out, Functional Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Activation Assay, Staining, Derivative Assay, Immunofluorescence, Hot Plate Test, Control

Journal: Bone Research

Article Title: TGF-β-induced fibrotic scar formation limits recovery of spinal cord injury

doi: 10.1038/s41413-026-00507-7

Figure Lengend Snippet: Conditional knockout of TGF-β type 2 receptor ( Tgfbr2 ) in pericytes reduces fibrotic scar formation in SCI mice. a Schematic diagram of Tgfbr2 knockout in Glast-Cre + pericytes. b Representative images of immunofluorescent analysis of PGP9.5 + (red) nerve fibers, fibronectin + (green) fibrotic scar, and DAPI (blue) staining of nuclei at 4 weeks after SCI. Scale bar, 200 µm. c , d Quantitative analysis of the intensity value of PGP9.5 and fibronectin (* P < 0.05, n = 6). e Representative images of immunofluorescent analysis of nerve axon–specific β-III-tubulin + (green), collagen III + (red) fibrotic scar, and DAPI (blue) staining of nuclei at 4 weeks after SCI. Scale bar, 200 µm. Right images are high resolution versions of the boxed regions in the left images. Scale bar, 50 µm. f Quantitative analysis of the intensity value of collagen III (* P < 0.05, n = 6). g Representative images of immunofluorescent analysis of neurotransmitter marker 5-HT + (red), nerve axon–specific β-III-tubulin + (green), and DAPI (blue) staining of nuclei in spinal cord lesion site of T10 in Tgfbr2 flox/flox sham group mice, Tgfbr2 flox/flox control mice, and Tgfbr2 Glast-creER −/− mice at 4 weeks after SCI. Scale bar, 200 µm. Right images are high-resolution versions of the boxed regions in the left images. Scale bar, 50 µm. h , i Quantitative analysis of the intensity value of 5-HT and β-III-tubulin (* P < 0.05, n = 6). j Representative images of immunofluorescent analysis of pSmad2 + (red), PDGFR-β + (green) pericytes and DAPI (blue) staining of nuclei at 7 days after SCI. Scale bar, 10 µm. k , l Quantitative analysis of the intensity mean value of PDGFR-β and the number of pSmad2 + cells (*** P < 0.001, **** P < 0.000 1, n = 6). m Representative Western blots showing the activation of pSmad signaling. Statistical significance was determined by multifactorial ANOVA, and all data are shown as means ± standard deviations. Source data are provided as a Source Data file

Article Snippet: We used antibodies recognizing mouse pSmad2 (1:1 000, Cell Signaling Technology, Inc., Danvers, MA, USA) and Smad2 (1:1 000, Cell Signaling Technology, Inc.) to examine the protein concentrations in the lysates.

Techniques: Knock-Out, Staining, Marker, Control, Western Blot, Activation Assay

Journal: Bone Research

Article Title: TGF-β-induced fibrotic scar formation limits recovery of spinal cord injury

doi: 10.1038/s41413-026-00507-7

Figure Lengend Snippet: Neonatal mice completely recover from SCI without scarring. a Schematic diagram illustrating the timeline of the experimental procedures. b Representative images of immunofluorescent analysis of pSmad2 (red), PDGFR-β (green), and DAPI (blue) in the spinal cord lesion site in neonatal mice on days 2, 7, and 12 with or without SCI. Scale bar, 50 µm. Right images are high-resolution versions of the boxed regions in the left images. Scale bar, 50 µm. c , d Quantitative analysis of the number of pSmad2 + cells and PDGFR-β + cells (** P < 0.01, *** P < 0.001, **** P < 0.000 1, n = 6). e Representative images of immunofluorescent analysis of β-III-tubulin (green), collagen III (red), and DAPI (blue) in the spinal cord lesion site in neonatal mice on days 2 and 12, and adult mice with or without SCI. Scale bar, 200 µm. f , g Quantitative analysis of the intensity mean value of collagen III + fibrotic scar and β-III-tubulin + nerves (* P < 0.05, **** P < 0.000 1, n = 6). Statistical significance was determined by multifactorial ANOVA, and all data are shown as means ± standard deviations. Source data are provided as a Source Data file

Article Snippet: We used antibodies recognizing mouse pSmad2 (1:1 000, Cell Signaling Technology, Inc., Danvers, MA, USA) and Smad2 (1:1 000, Cell Signaling Technology, Inc.) to examine the protein concentrations in the lysates.

Techniques:

Journal: eLife

Article Title: Activin A marks a novel progenitor cell population during fracture healing and reveals a therapeutic strategy

doi: 10.7554/elife.89822

Figure Lengend Snippet: Figure 6. Local Activin A implantation promotes fracture healing. (A) Representative Safranin O/Fast green staining histochemical images of fracture calluses at days 5, 14, and 28 post fracture. Two-month-old (2M) or 20-month-old (20M) mice were implanted with a 50-μl Matrigel aliquot containing vehicle (Veh) or Activin A (ACT) (1 μg) at the fracture site immediately after surgery. Scale bar, 1 mm. (B) Callus area, cartilage area, and bone area were quantified at indicated time points. n = 4 mice/group. (C) Immunofluorescence images of pSMAD2 and αSMA in fracture calluses of control and

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) Col2a1- Cre Jackson Laboratory Stock #: 003554 Genetic reagent (Mus musculus) Rosa26LSL- tdTomato Jackson Laboratory Stock #: 007909 Genetic reagent (Mus musculus) C57BL/6 Jackson Laboratory Stock #: 000664 Antibody Mouse monoclonal neutralizing antibody against Activin A Biolegend Cat #: 693604 10 mg/kg Antibody Rabbit monoclonal anti- mouse pSMAD2 Cell Signaling Cat #: 3108S 1:200 Antibody Mouse monoclonal anti- mouse αSMA Sigma Cat #: A5228 1:200 Yao et al. eLife 2023;12:e89822.

Techniques: Staining, Immunofluorescence, Control

Journal: eLife

Article Title: Activin A marks a novel progenitor cell population during fracture healing and reveals a therapeutic strategy

doi: 10.7554/eLife.89822

Figure Lengend Snippet: ( A ) Representative Safranin O/Fast green staining images of fracture calluses at days 5, 7, 10, and 14 post fracture. Mice received subcutaneous injections of control IgG2b isotype or neutralizing monoclonal antibody against Activin A (nActA.AB, 10 mg/kg) twice a week after fracture. Scale bar, 1 mm. ( B ) Callus area, cartilage area, and bone area were quantified at indicated time points. n = 4–7 mice/time point. ( C ) Measurement of fracture healing scores at 6 weeks post fracture. n = 10 mice/group. ( D ) Mechanical testing was performed on bones at 6 weeks post fracture. n = 10 mice/group. ( E ) Immunofluorescence images of pSMAD2 and αSMA in fracture calluses of control (isotype) and nActA.AB-treated mice at day 7 post fracture. White arrows point to pSMAD2+αSMA+ cells and yellow arrows point to pSMAD2+αSMA− cells. Scale bar, 500 μm (low mag), 50 μm (high mag). ( F ) Percentages of pSMAD2+ and αSMA+ cells in fracture calluses and pSMAD2+ cells within the αSMA+/− populations were quantified. n = 3 mice/group. ( G ) qRT-PCR analyses of Acta2 and Inhba expression in day 7 callus from 2-month-old mice treated with nActA.AB versus isotype control. n = 4 mice/group. Data are expressed as means ± SD and analyzed by unpaired two-tailed t-test. Figure 5—source data 1. Source data for . Figure 5—source data 2. Source data for . Figure 5—source data 3. Source data for . Figure 5—source data 4. Source data for . Figure 5—source data 5. Source data for .

Article Snippet: Antibody , Rabbit monoclonal anti-mouse pSMAD2 , Cell Signaling , Cat #: 3108S , 1:200.

Techniques: Staining, Control, Immunofluorescence, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: eLife

Article Title: Activin A marks a novel progenitor cell population during fracture healing and reveals a therapeutic strategy

doi: 10.7554/eLife.89822

Figure Lengend Snippet: ( A ) Representative Safranin O/Fast green staining histochemical images of fracture calluses at days 5, 14, and 28 post fracture. Two-month-old (2M) or 20-month-old (20M) mice were implanted with a 50-μl Matrigel aliquot containing vehicle (Veh) or Activin A (ACT) (1 μg) at the fracture site immediately after surgery. Scale bar, 1 mm. ( B ) Callus area, cartilage area, and bone area were quantified at indicated time points. n = 4 mice/group. ( C ) Immunofluorescence images of pSMAD2 and αSMA in fracture calluses of control and ACT-implanted mice at day 5 post fracture. White arrows point to pSMAD2+αSMA+ cells and yellow arrows point to pSMAD2+αSMA− cells. Scale bar, 500 μm (low mag), 50 μm (high mag). ( D ) Percentage of pSMAD2+, αSMA+ cells in fracture calluses and pSMAD2+ cells within αSMA+/− populations were quantified. n = 3 mice/group. ( E ) Fracture healing scores were quantified in bones of 2- and 20-month-old mice at 6 weeks post fracture. n = 10 mice/group. ( F ) Mechanical testing was performed on bones of 2- and 20-month-old mice at 6 weeks post fracture. n = 10 mice/group. Data are expressed as means ± SD and analyzed by unpaired two-tailed t-test. Figure 6—source data 1. Source data for . Figure 6—source data 2. Source data for . Figure 6—source data 3. Source data for . Figure 6—source data 4. Source data for .

Article Snippet: Antibody , Rabbit monoclonal anti-mouse pSMAD2 , Cell Signaling , Cat #: 3108S , 1:200.

Techniques: Staining, Immunofluorescence, Control, Two Tailed Test

Journal: eLife

Article Title: Activin A marks a novel progenitor cell population during fracture healing and reveals a therapeutic strategy

doi: 10.7554/eLife.89822

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti-mouse pSMAD2 , Cell Signaling , Cat #: 3108S , 1:200.

Techniques: Flow Cytometry, Recombinant, Software

Journal: International Journal of Molecular Sciences

Article Title: Leucine-Rich Alpha-2 Glycoprotein 1 Accumulates in Complicated Atherosclerosis and Promotes Calcification

doi: 10.3390/ijms242216537

Figure Lengend Snippet: Effect of LRG1 on TGFβ-induced SMAD signaling. ( A , B ) Quantification and representative capillary Western blots of pSMAD1-5/SMAD1 and ( B ) pSMAD2-3/SMAD2 in MOVAS treated with 20 μg/mL LRG1 with or without 10 ng/mL TGFβ for 1 h. n = 3–5 independent experiments/group; ns: non significative; ** p < 0.01 via Mann–Whitney U test. Data are presented as median with lower and upper quartiles.

Article Snippet: Proteins were loaded into Wes capillaries, and columns were probed with anti-mouse antibodies for pSMAD2 (diluted 20 e with samples diluted at 1⁄4, 3108 Cell signaling), SMAD2/3 (diluted 100 e with samples undiluted, 610,842 BD Science), pSMAD1/5 (diluted 50 e with samples undiluted, 9516 Cell signaling), SMAD1 (diluted 100 e with samples diluted at 1⁄2, 9743 Cell signaling).

Techniques: Western Blot, MANN-WHITNEY