smad3 ser423 425 rabbit mab  (Cell Signaling Technology Inc)

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

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.

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

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.

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

Journal: Science Advances

Article Title: Jagged1 regulates extracellular matrix deposition and remodeling in triple-negative breast cancer

doi: 10.1126/sciadv.aea9562

Figure Lengend Snippet: ( A ) Tumor masses of MDA-MB-231 Jag1WT or Jag1KO cell cocultures with MEF cells in the chick CAM xenograft model. P values are calculated by a two-tailed unpaired t test. n = 37 tumors for Jag1WT and n = 40 for Jag1KO. ( B ) Hematoxylin and eosin–stained tissue sections of CAM tumors in (A). Scale bars, 100 μm. CAM tumor cryosections stained for ( C ) collagen (CNA35; green in merge), FN (magenta in merge), actin (phalloidin; orange in merge), and nuclei (DAPI; blue in merge); ( D ) phosphorylated SMAD2 (Ser 465/467 )/SMAD3 (Ser 423/425 ) (p-SMAD); and ( E ) collagen (CNA35; green in merge), pan-cytokeratin (pan-cKRT; magenta in merge), αSMA (orange in merge), and nuclei (DAPI; blue in merge). Scale bars, 500 μm. ( F ) Quantifications of αSMA, p-SMAD, CNA35, and FN fluorescence integrated densities in CAM tumor cryosections. n = 4 tumors for αSMA and p-SMAD and n = 3 tumors for CNA35 and FN in each group. αSMA was quantified from glandular structures containing fibroblasts, and CNA35, FN, and p-SMAD were quantified from the whole-tumor area, excluding possible surrounding CAM. Data are presented as the means ± SEM. P values are calculated by a two-tailed unpaired t test. ( G ) mRNA expression level correlation with Jag1 mRNA expression in patients with breast cancer (TCGA, Cell 2015, n = 817) and ( H ) relapse-free survival of patients with low (black) or high (red) expression of ACTA2 , FN1 , COL1A2 , or COL3A1 in all breast cancer cases and in basal breast cancer separately in the Kaplan-Meier Plotter database .

Article Snippet: The following antibodies and dilutions were used: 1:1000 Jagged1 (Cell Signaling Technology, cat. no. 2620, RRID: AB_10693295), 1:1000 phospho-Smad2 (Ser 465/467 )/Smad3 (Ser 423/425 ) (Cell Signaling Technology, cat. no. 8828, RRID: AB_2631089), 1:1000 Smad2/3 (Santa Cruz Biotechnology, cat. no. sc-133098, RRID: AB_2193048), 1:1000 αSMA (Cell Signaling Technology, cat. no. 19245, RRID: AB_2734735), 1:2500 FN (BD Biosciences, cat. no. 610077, RRID: AB_2105706), 1:1000 ERα (Santa Cruz Biotechnology, cat. no. sc-8002, RRID: AB_627558), 1:1000 β-tubulin (Cell Signaling Technology, cat. no. 86298, RRID: AB_2715541), 1:200,000 β-actin (Sigma-Aldrich, cat. no. A1978, RRID: AB_4766922), and 1:5000 HSC70 (Enzo Life Sciences, cat. no. ADI-SPA-815, RRID: AB_10617277).

Techniques: Two Tailed Test, Staining, Fluorescence, Expressing