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Cell Signaling Technology Inc anti p smad2
Anti P Smad2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1773 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/phospho+smad2/Phospho-Smad2+(Ser465%2F467)+Rabbit+mAb/pmc12914543-97-104-106
Average 96 stars, based on 1773 article reviews

anti p smad2 - by Bioz Stars, 2026-10

96/100 stars

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Article Title: Assessing the clinical significance of a novel rare variant in Loeys-Dietz Syndrome by combining AI-driven modelling and cell biology
Article Snippet: Protein concentrations were determined using the Thermo ScientificTM PierceTM BCA Protein Assay Kits (Thermo Fisher Scientific), and equal amounts of protein (10 μg per sample) were denatured, resolved on 4–12% Bis-Tris polyacrylamide gels (NuPAGETM, Thermo Fisher Scientific), and transferred onto nitrocellulose membranes. .. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) for 1 h at room temperature, followed by overnight incubation at 4 °C with the following primary antibodies: TGF-β Receptor II (E5M6F) (1:1,000; Cell Signaling #41896), total SMAD2/3 (1:1,000; Cell Signaling, #8685), phospho-SMAD2 (1:1,000; Cell Signaling, #3108), PARP1 (E102) (1:5,000; Abcam, #32138), and GAPDH (1:1,000; Santa Cruz Biotechnology, sc-32233). ..

Incubation:

Article Title: Molecular Engineering of the Helminth TGF ‐β Mimetics, TGM1 and TGM4, Reveals a Novel Antagonist of TGF ‐β Signaling in Fibroblasts
Article Snippet: Briefly, following ligand treatment, cell lysates were analyzed on 4%–12% bis‐tris SDS ‐ PAGE gels and transferred onto a nitrocellulose membrane using iBlot2 (Invitrogen, IB21001 ). .. Membranes were treated in 5% non‐fat milk blocking solution for 1 h and incubated with primary rabbit polyclonal anti‐ SMAD2 /3 (Cell Signaling Technology #3102, #5678), or with rabbit monoclonal antibody D27F4 to phospho‐ SMAD2 (Ser465/467)/ SMAD3 (Ser423/425) rabbit mAb (Cell Signaling Technology, Cat. No. 8828), each at 1:1000 in 5% BSA containing TBST , overnight at 4°C. .. Following three 5‐min washes with TBST , fluorescent‐conjugated secondary polyclonal goat anti‐rabbit IgG ( DyLight 680, Invitrogen SA535571 ) diluted 1:10 000 in 5% BSA containing TBST was used to detect binding by the Odyssey CLx Imaging System ( LI ‐ COR Biosciences).

Article Title: SIK1 drives lipid-induced insulin resistance in skeletal muscle by linking TGFβ1-Smad2/3 activation to PDE4-cAMP dysregulation.
Article Snippet: Excessive lipid accumulation in skeletal muscle contributes to insulin resistance.. Salt-inducible kinase 1 (SIK1) is known to be involved in myogenic differentiation, yet its role in lipid-induced skeletal muscle insulin resistance remains unclear.. Here, we identified the functional role of SIK1 in skeletal muscle insulin resistance under lipid overload and delineated the underlying signaling mechanisms.

Article Title: Jagged1 regulates extracellular matrix deposition and remodeling in triple-negative breast cancer
Article Snippet: .. Primary antibodies against FN (BD Biosciences, cat. no. 610077, RRID: AB_2105706), αSMA (Cell Signaling Technology, cat. no. 19245, RRID: AB_2734735), pan-cytokeratin (Sigma-Aldrich, cat. no. C2562, RRID: AB_476839), phospho-Smad2 (Ser 465/467 )/Smad3 (Ser 423/425 ) (Cell Signaling Technology, cat. no. 8828, RRID: AB_2631089), and desmin (Bio SB, cat. no. BSB 5460, RRID: AB_3716523) were diluted 1:100 in blocking buffer and incubated on cryosections overnight at +4°C. .. The following day, cryosections were washed three times with PBST and incubated with secondary antibodies (4 μg/ml; anti-mouse Alexa Fluor 555, Molecular Probes, cat. no. A-21424, RRID: AB_141780; anti-rabbit Alexa Fluor 647, Molecular Probes, cat. no. A-31573, RRID: AB_2536183), 165 nM Alexa Fluor 633 Phalloidin (Invitrogen, no. A22284), CNA35 (20 ng/μl), and 600 nM DAPI in blocking buffer for 1 hour at RT.

Blocking Assay:

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ECM-related cues promote RepC-associated gene expression and activate TGF-β/SMAD signaling in human articular chondrocytes. (A) Relative mRNA expression levels of RepC-associated genes (CILP, OGN, MMP13, and FRZB) in primary human articular chondrocytes treated with TGF-β1 (5 ng/mL), FN1 (10 μg/mL), or their combination. Gene expression was normalized to internal controls and expressed relative to untreated controls (Con). (B) Quantification of protein expression levels of CILP and MMP13, as well as phosphorylation levels of SMAD2 and SMAD3, under the same treatment conditions as in (A) . Protein levels were normalized to β-actin, and phosphorylation levels were normalized to total SMAD2 or SMAD3, respectively. (C) Representative immunoblot images showing CILP, MMP13, phosphorylated SMAD2 (P-SMAD2), total SMAD2, phosphorylated SMAD3 (P-SMAD3), total SMAD3, and β-actin under control, TGF-β1, FN1, and combined TGF-β1 + FN1 stimulation conditions. Data are presented as mean ± SD from n = 3 independent biological replicates. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc multiple-comparison test. **p < 0.01, ***p < 0.001.

Journal: Frontiers in Endocrinology

Article Title: ECM remodeling features in reparative chondrocytes during knee osteoarthritis

doi: 10.3389/fendo.2026.1773139

Figure Lengend Snippet: ECM-related cues promote RepC-associated gene expression and activate TGF-β/SMAD signaling in human articular chondrocytes. (A) Relative mRNA expression levels of RepC-associated genes (CILP, OGN, MMP13, and FRZB) in primary human articular chondrocytes treated with TGF-β1 (5 ng/mL), FN1 (10 μg/mL), or their combination. Gene expression was normalized to internal controls and expressed relative to untreated controls (Con). (B) Quantification of protein expression levels of CILP and MMP13, as well as phosphorylation levels of SMAD2 and SMAD3, under the same treatment conditions as in (A) . Protein levels were normalized to β-actin, and phosphorylation levels were normalized to total SMAD2 or SMAD3, respectively. (C) Representative immunoblot images showing CILP, MMP13, phosphorylated SMAD2 (P-SMAD2), total SMAD2, phosphorylated SMAD3 (P-SMAD3), total SMAD3, and β-actin under control, TGF-β1, FN1, and combined TGF-β1 + FN1 stimulation conditions. Data are presented as mean ± SD from n = 3 independent biological replicates. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc multiple-comparison test. **p < 0.01, ***p < 0.001.

Article Snippet: The primary antibodies included CILP (PA5-18553, Thermo Fisher Scientific; 1:1500), MMP13 (18165-1-AP, Proteintech; 1:1000), phospho-SMAD2 (ET1612-32, HUABIO; 1:5000), phospho-SMAD3 (ET1609-41, HUABIO; 1:5000), SMAD2 (12570-1-AP, Proteintech; 1:2000), SMAD3 (66516-1-Ig, Proteintech; 1:2000), and β-actin (66009-1-Ig, Proteintech; 1:20000).

Techniques: Gene Expression, Expressing, Phospho-proteomics, Western Blot, Control, Comparison

Canonical TGF-β signalling pathway. TGF-β ligand binds to the type II receptor (TGFBR2), which recruits and phosphorylates the type I receptor (TGFBR1). Activated TGFBR1 phosphorylates receptor-regulated SMADs (SMAD2/3), which form a complex with the common mediator SMAD4. This complex translocates to the nucleus, where it regulates the transcription of target genes controlling proliferation, differentiation, apoptosis, and extracellular matrix homeostasis

Journal: medRxiv

Article Title: Assessing the clinical significance of a novel rare variant in Loeys-Dietz Syndrome by combining AI-driven modelling and cell biology

doi: 10.64898/2026.03.30.26349510

Figure Lengend Snippet: Canonical TGF-β signalling pathway. TGF-β ligand binds to the type II receptor (TGFBR2), which recruits and phosphorylates the type I receptor (TGFBR1). Activated TGFBR1 phosphorylates receptor-regulated SMADs (SMAD2/3), which form a complex with the common mediator SMAD4. This complex translocates to the nucleus, where it regulates the transcription of target genes controlling proliferation, differentiation, apoptosis, and extracellular matrix homeostasis

Article Snippet: Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) for 1 h at room temperature, followed by overnight incubation at 4 °C with the following primary antibodies: TGF-β Receptor II (E5M6F) (1:1,000; Cell Signaling #41896), total SMAD2/3 (1:1,000; Cell Signaling, #8685), phospho-SMAD2 (1:1,000; Cell Signaling, #3108), PARP1 (E102) (1:5,000; Abcam, #32138), and GAPDH (1:1,000; Santa Cruz Biotechnology, sc-32233).

Techniques:

TGFBR2 E431K disrupts the canonical TGF-β signalling pathway in vitro. HEK293T cells were transiently transfected with 0.5 μg of pcDNA3.1+ empty vector, WT TGFBR2, or TGFBR2 E431K for 48 h and stimulated with TGF-β1 (10 ng/mL). Western blotting was used to assess (a) TGFBR2 abundance and SMAD pathway activation, including (b) whole-cell pSMAD2 and total SMAD2/3 and (c) nuclear pSMAD2 at 1 h post-stimulation, as well as (d) pSMAD2 levels at 1, 6, and 9 h post-stimulation. Downstream transcriptional responses ( SERPINE1 , COL1A1 ) were quantified by RT–qPCR. All experiments were performed in triplicate; Data are shown as mean ± Standard error of the mean (SEM), with statistical significance of *p < 0.05 , **p < 0.01 , ***p < 0.001 , and ****p < 0.0001

Journal: medRxiv

Article Title: Assessing the clinical significance of a novel rare variant in Loeys-Dietz Syndrome by combining AI-driven modelling and cell biology

doi: 10.64898/2026.03.30.26349510

Figure Lengend Snippet: TGFBR2 E431K disrupts the canonical TGF-β signalling pathway in vitro. HEK293T cells were transiently transfected with 0.5 μg of pcDNA3.1+ empty vector, WT TGFBR2, or TGFBR2 E431K for 48 h and stimulated with TGF-β1 (10 ng/mL). Western blotting was used to assess (a) TGFBR2 abundance and SMAD pathway activation, including (b) whole-cell pSMAD2 and total SMAD2/3 and (c) nuclear pSMAD2 at 1 h post-stimulation, as well as (d) pSMAD2 levels at 1, 6, and 9 h post-stimulation. Downstream transcriptional responses ( SERPINE1 , COL1A1 ) were quantified by RT–qPCR. All experiments were performed in triplicate; Data are shown as mean ± Standard error of the mean (SEM), with statistical significance of *p < 0.05 , **p < 0.01 , ***p < 0.001 , and ****p < 0.0001

Techniques: In Vitro, Transfection, Plasmid Preparation, Western Blot, Activation Assay, Quantitative RT-PCR

Inhibition of TGM1 and TGF‐β Signaling by TGM4. (a) Schematic representation of the 5 domains of TGM1 and TGM4, indicating amino acid lengths, receptor specificity, and percentage amino acid identity between the two proteins in each domain. (b) Inhibition of TGF‐β (5 ng/mL) and TGM1 (10 ng/mL) signaling in MFB‐F11 transcriptional reporter murine fibroblasts by increasing concentrations of TGM4, as measured by the release of alkaline phosphatase. Data are means ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Dunnett's multiple comparison test; showing statistics comparing TGF‐β responses with TGM4 versus no TGM4. **** p < 0.0001. (c) Inhibition of SMAD2/3 phosphorylation in MFB‐F11 fibroblasts as measured by Western blotting of cell lysates following stimulation with TGF‐β and/or TGM4 (2.5 ng/mL) as indicated. Data are from one of three independent experiments. α‐Tubulin, loading control. (d) Inhibition of signaling in murine NIH 3 T3 fibroblasts expressing dynGFP under a CAGA promoter responsive to SMAD3 signaling, following stimulation with TGF‐β (1 ng/mL) or TGM1 (2.5 ng/mL) in the presence of the indicated doses of TGM4. Data are means ± SD, n = 3 from 1 of three replicate experiments, analyzed by unpaired t test. *** p < 0.001, **** p < 0.0001. (e) Kinetics of inhibition of 5 ng/mL TGF‐β signaling in MFB‐F11 transcriptional reporter fibroblasts exposed to 100 ng/mL TGM4 or the pharmacological TGFBR1‐like kinase inhibitor SB431542 (5 μM) at different time points relative to administration of TGF‐β. Data are mean ± SD, n = 3 from one of three replicate experiments analyzed by two‐way ANOVA with Sidak's multiple comparison test; showing statistics comparing TGF‐β responses with TGM4 versus no TGM4. **** p < 0.0001; ns, not significant ( p > 0.05). (f and g) Inhibition of signaling in MFB‐F11 transcriptional reporter fibroblasts receiving the indicated doses of TGM4 at 30 min prior to 5 ng/mL TGF‐β or 10 ng/mL TGM1, without (f) and with (g) washing and removal of TGM4 at the indicated doses. Data are mean ± SD, n = 3 from one of three replicate experiments, analyzed by two‐way ANOVA with Dunnett's multiple comparison test; showing statistics comparing TGM1 or TGF‐β responses with TGM4 versus no TGM4. * p < 0.05, ** p < 0.01, **** p < 0.0001; ns, not significant ( p > 0.05). (h and i) TGM4 treatment of CD44 KO MFB‐F11 transcriptional reporter cells (h) does not inhibit TGF‐β signaling, whereas it is highly inhibitory in WT cells (i); cells were stimulated with 5 ng/mL TGF‐β or 10 ng/mL TGM1. Data are mean ± SD, n = 3 from one of three replicate experiments, analyzed by two‐way ANOVA with Dunnett's multiple comparison test; showing statistics comparing TGM1 or TGF‐β responses with TGM4 versus no TGM4. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant ( p > 0.05).

Journal: The FASEB Journal

Article Title: Molecular Engineering of the Helminth TGF ‐β Mimetics, TGM1 and TGM4, Reveals a Novel Antagonist of TGF ‐β Signaling in Fibroblasts

doi: 10.1096/fj.202503194R

Figure Lengend Snippet: Inhibition of TGM1 and TGF‐β Signaling by TGM4. (a) Schematic representation of the 5 domains of TGM1 and TGM4, indicating amino acid lengths, receptor specificity, and percentage amino acid identity between the two proteins in each domain. (b) Inhibition of TGF‐β (5 ng/mL) and TGM1 (10 ng/mL) signaling in MFB‐F11 transcriptional reporter murine fibroblasts by increasing concentrations of TGM4, as measured by the release of alkaline phosphatase. Data are means ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Dunnett's multiple comparison test; showing statistics comparing TGF‐β responses with TGM4 versus no TGM4. **** p < 0.0001. (c) Inhibition of SMAD2/3 phosphorylation in MFB‐F11 fibroblasts as measured by Western blotting of cell lysates following stimulation with TGF‐β and/or TGM4 (2.5 ng/mL) as indicated. Data are from one of three independent experiments. α‐Tubulin, loading control. (d) Inhibition of signaling in murine NIH 3 T3 fibroblasts expressing dynGFP under a CAGA promoter responsive to SMAD3 signaling, following stimulation with TGF‐β (1 ng/mL) or TGM1 (2.5 ng/mL) in the presence of the indicated doses of TGM4. Data are means ± SD, n = 3 from 1 of three replicate experiments, analyzed by unpaired t test. *** p < 0.001, **** p < 0.0001. (e) Kinetics of inhibition of 5 ng/mL TGF‐β signaling in MFB‐F11 transcriptional reporter fibroblasts exposed to 100 ng/mL TGM4 or the pharmacological TGFBR1‐like kinase inhibitor SB431542 (5 μM) at different time points relative to administration of TGF‐β. Data are mean ± SD, n = 3 from one of three replicate experiments analyzed by two‐way ANOVA with Sidak's multiple comparison test; showing statistics comparing TGF‐β responses with TGM4 versus no TGM4. **** p < 0.0001; ns, not significant ( p > 0.05). (f and g) Inhibition of signaling in MFB‐F11 transcriptional reporter fibroblasts receiving the indicated doses of TGM4 at 30 min prior to 5 ng/mL TGF‐β or 10 ng/mL TGM1, without (f) and with (g) washing and removal of TGM4 at the indicated doses. Data are mean ± SD, n = 3 from one of three replicate experiments, analyzed by two‐way ANOVA with Dunnett's multiple comparison test; showing statistics comparing TGM1 or TGF‐β responses with TGM4 versus no TGM4. * p < 0.05, ** p < 0.01, **** p < 0.0001; ns, not significant ( p > 0.05). (h and i) TGM4 treatment of CD44 KO MFB‐F11 transcriptional reporter cells (h) does not inhibit TGF‐β signaling, whereas it is highly inhibitory in WT cells (i); cells were stimulated with 5 ng/mL TGF‐β or 10 ng/mL TGM1. Data are mean ± SD, n = 3 from one of three replicate experiments, analyzed by two‐way ANOVA with Dunnett's multiple comparison test; showing statistics comparing TGM1 or TGF‐β responses with TGM4 versus no TGM4. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant ( p > 0.05).

Article Snippet: Membranes were treated in 5% non‐fat milk blocking solution for 1 h and incubated with primary rabbit polyclonal anti‐ SMAD2 /3 (Cell Signaling Technology #3102, #5678), or with rabbit monoclonal antibody D27F4 to phospho‐ SMAD2 (Ser465/467)/ SMAD3 (Ser423/425) rabbit mAb (Cell Signaling Technology, Cat. No. 8828), each at 1:1000 in 5% BSA containing TBST , overnight at 4°C.

Techniques: Inhibition, Comparison, Phospho-proteomics, Western Blot, Control, Expressing

Dimerization of TGM4 enhances activation of fibroblasts and T cells. Molar equivalents used were based on monomeric ligand mol.wt of 49.1 kDa and Fc‐dimerized ligand mol.wt. of 144.7 kDa (Table S2), giving a ratio of 49.1:72.4, or 1.47 ng of Fc dimer per 1 ng of monomer. (a) TGM1‐Fc dimerization does not enhance its ability to activate SMAD3 transcriptional response in MFB‐F11 fibroblasts. Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM1 monomer and Fc dimer. *** p < 0.001; **** p < 0.0001; ns, not significant ( p > 0.05). (b) TGM4‐Fc dimer activates SMAD3 transcriptional response in MFB‐F11 fibroblasts. Data are mean ± SD, n = 3 from one of three replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM4 monomer and dimer. **** p < 0.0001. (c) SMAD3 transcriptional response assay in MFB‐F11 fibroblasts comparing monomeric TGM4 and TGM4‐Fc‐dimer, either alone or in addition to TGM1. Data are mean ± SD, n = 3 s, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM4 monomer and dimer. **** p < 0.0001. (d) TGM4‐Fc dimer activation of SMAD3 transcriptional response is dependent on CD44 expression in MFB‐F11 cells. CD44 KO cells were constructed, as described previously . Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM4 dimer responses in WT and CD44KO cells. **** p < 0.0001. (e) TGM4 D1‐3 dimer is unable to activate SMAD3 transcriptional response in MFB‐F11 fibroblasts in the same manner as full‐length TGM4 dimer. Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM4 full‐length and D1‐3 dimers. **** p < 0.0001. (f) TGM4 induction of Foxp3 expression in murine spleen CD4 + T cells by TGM4 in monomeric and dimeric forms. Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test. **** p < 0.0001. (g and h) SMAD2/3 phosphorylation of murine Foxp3 + (g) and Foxp3 − (h) T cells as analyzed by flow cytometry 1–16 h post‐stimulation with 100 ng/mL TGM1 or TGM4, or 139 ng/mL of TGM4‐Fc‐dimer (representing molar equivalent for binding sites). Data are mean ± SD, n = 2 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test. * p < 0.05; ** p < 0.01;**** p < 0.0001.

Journal: The FASEB Journal

Article Title: Molecular Engineering of the Helminth TGF ‐β Mimetics, TGM1 and TGM4, Reveals a Novel Antagonist of TGF ‐β Signaling in Fibroblasts

doi: 10.1096/fj.202503194R

Figure Lengend Snippet: Dimerization of TGM4 enhances activation of fibroblasts and T cells. Molar equivalents used were based on monomeric ligand mol.wt of 49.1 kDa and Fc‐dimerized ligand mol.wt. of 144.7 kDa (Table S2), giving a ratio of 49.1:72.4, or 1.47 ng of Fc dimer per 1 ng of monomer. (a) TGM1‐Fc dimerization does not enhance its ability to activate SMAD3 transcriptional response in MFB‐F11 fibroblasts. Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM1 monomer and Fc dimer. *** p < 0.001; **** p < 0.0001; ns, not significant ( p > 0.05). (b) TGM4‐Fc dimer activates SMAD3 transcriptional response in MFB‐F11 fibroblasts. Data are mean ± SD, n = 3 from one of three replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM4 monomer and dimer. **** p < 0.0001. (c) SMAD3 transcriptional response assay in MFB‐F11 fibroblasts comparing monomeric TGM4 and TGM4‐Fc‐dimer, either alone or in addition to TGM1. Data are mean ± SD, n = 3 s, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM4 monomer and dimer. **** p < 0.0001. (d) TGM4‐Fc dimer activation of SMAD3 transcriptional response is dependent on CD44 expression in MFB‐F11 cells. CD44 KO cells were constructed, as described previously . Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM4 dimer responses in WT and CD44KO cells. **** p < 0.0001. (e) TGM4 D1‐3 dimer is unable to activate SMAD3 transcriptional response in MFB‐F11 fibroblasts in the same manner as full‐length TGM4 dimer. Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test, showing statistics comparing TGM4 full‐length and D1‐3 dimers. **** p < 0.0001. (f) TGM4 induction of Foxp3 expression in murine spleen CD4 + T cells by TGM4 in monomeric and dimeric forms. Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test. **** p < 0.0001. (g and h) SMAD2/3 phosphorylation of murine Foxp3 + (g) and Foxp3 − (h) T cells as analyzed by flow cytometry 1–16 h post‐stimulation with 100 ng/mL TGM1 or TGM4, or 139 ng/mL of TGM4‐Fc‐dimer (representing molar equivalent for binding sites). Data are mean ± SD, n = 2 from one of two replicate experiments, analyzed by two‐way ANOVA with Sidák's multiple comparison test. * p < 0.05; ** p < 0.01;**** p < 0.0001.

Techniques: Activation Assay, Comparison, Expressing, Construct, Phospho-proteomics, Flow Cytometry, Binding Assay

Activation of fibroblast signaling by domain swap constructs. (a–c) Analysis of TGM4 D3 (a), D45 (b), and D3‐5 (c) domain exchanges, assayed for activation of SMAD3 transcriptional response in MFB‐F11 fibroblasts, as measured by release of alkaline phosphatase. In each case, the chimeric protein is shown in yellow. Data are mean ± SD, n = 3 from one of two replicate experiments, with comparisons of TGM1 with the indicated chimera analyzed by two‐way ANOVA with Sidák's multiple comparison test. ** p < 0.01;**** p < 0.0001; ns, not significant ( p > 0.05). (d–f) Analysis of TGM1 D3 (d), D45 (e), and D3‐5 (f) domain exchanges, assayed for activation of MFB‐F11fibroblasts. In each case, the chimeric protein is shown in yellow. Data are mean ± SD, n = 3 from one of two replicate experiments, with comparisons of TGM1 with the indicated chimera analyzed by two‐way ANOVA with Sidák's multiple comparison test. **** p < 0.0001. (g) Analysis of Foxp3 expression in mouse splenic T cells, cultured in anti‐CD3‐coated wells and incubated with 400 U/mL of IL‐2 and 100 ng/mL of the indicated ligands. Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001. (h) Western blot analysis of phospho‐SMAD (pSMAD) levels in lysates of RAW246.7 macrophages that were challenged by the indicated chimeric proteins, following incubation with 100 ng/mL of ligand for 1 h. α‐Tubulin, loading control. (i and j) Flow cytometric analyses of anti‐CD44 binding to small intestinal CD4 + T cells were (CD45 + , CD3 + CD4 + CD19 − ), macrophages (CD45 + CD11b + CD64 + Ly6C + MHC‐II + ), and fibroblasts (CD45 − CD31 − ESAM − Podoplanin + CD4 + ), presented as percentage positive and MFI (i) and as histograms (j). MFI, Mean Fluorescence Intensity; FMO, Fluorescence Minus One, that is, in the absence of anti‐CD44 antibody. Data presented are from five individual C57BL/6 mice analyzed by one‐way ANOVA; ** p < 0.01; **** p < 0.0001.

Journal: The FASEB Journal

Article Title: Molecular Engineering of the Helminth TGF ‐β Mimetics, TGM1 and TGM4, Reveals a Novel Antagonist of TGF ‐β Signaling in Fibroblasts

doi: 10.1096/fj.202503194R

Figure Lengend Snippet: Activation of fibroblast signaling by domain swap constructs. (a–c) Analysis of TGM4 D3 (a), D45 (b), and D3‐5 (c) domain exchanges, assayed for activation of SMAD3 transcriptional response in MFB‐F11 fibroblasts, as measured by release of alkaline phosphatase. In each case, the chimeric protein is shown in yellow. Data are mean ± SD, n = 3 from one of two replicate experiments, with comparisons of TGM1 with the indicated chimera analyzed by two‐way ANOVA with Sidák's multiple comparison test. ** p < 0.01;**** p < 0.0001; ns, not significant ( p > 0.05). (d–f) Analysis of TGM1 D3 (d), D45 (e), and D3‐5 (f) domain exchanges, assayed for activation of MFB‐F11fibroblasts. In each case, the chimeric protein is shown in yellow. Data are mean ± SD, n = 3 from one of two replicate experiments, with comparisons of TGM1 with the indicated chimera analyzed by two‐way ANOVA with Sidák's multiple comparison test. **** p < 0.0001. (g) Analysis of Foxp3 expression in mouse splenic T cells, cultured in anti‐CD3‐coated wells and incubated with 400 U/mL of IL‐2 and 100 ng/mL of the indicated ligands. Data are mean ± SD, n = 3 from one of two replicate experiments, analyzed by one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001. (h) Western blot analysis of phospho‐SMAD (pSMAD) levels in lysates of RAW246.7 macrophages that were challenged by the indicated chimeric proteins, following incubation with 100 ng/mL of ligand for 1 h. α‐Tubulin, loading control. (i and j) Flow cytometric analyses of anti‐CD44 binding to small intestinal CD4 + T cells were (CD45 + , CD3 + CD4 + CD19 − ), macrophages (CD45 + CD11b + CD64 + Ly6C + MHC‐II + ), and fibroblasts (CD45 − CD31 − ESAM − Podoplanin + CD4 + ), presented as percentage positive and MFI (i) and as histograms (j). MFI, Mean Fluorescence Intensity; FMO, Fluorescence Minus One, that is, in the absence of anti‐CD44 antibody. Data presented are from five individual C57BL/6 mice analyzed by one‐way ANOVA; ** p < 0.01; **** p < 0.0001.

Techniques: Activation Assay, Construct, Comparison, Expressing, Cell Culture, Incubation, Western Blot, Control, Binding Assay, Fluorescence

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