recombinant human latency associated peptide lap transforming growth factor tgf β1  (R&D Systems)

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Hyperactive Transforming Growth Factor-β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

doi: 10.1002/jbmr.1992

Figure Lengend Snippet: Nf1 gene dose regulates TGF-β1 production by osteoblasts. (A) Serum TGF-β1 levels were measured by ELISA in WT and Nf1flox/−;Col2.3Cre mice. n = 4 mice per group. ****p < 0.0001. (B) TGF-β1 mRNA expression in WT, Nf1+/−, Nf1−/− osteoblast progenitors was examined by quantitative PCR. n = 3 technical replicates. The experiment was repeated twice with similar results using independent cell lines. **p < 0.01, ****p < 0.0001. (C) Protein extracts were examined by ELISA to determine TGF-β1 protein expression. n = 3 technical replicates. The assay was repeated twice with similar results using independent cell lines. **p < 0.01, ****p < 0.0001. (D) Serum TGF-β1 levels were measured by ELISA in human NF1 patients and healthy controls. n = 7 samples per group. **p < 0.01.

Article Snippet: Immunoblots were performed using antibodies specific to p-Smad2 (Cell Signaling), total Smad2 (Cell Signaling), active TGF-β1 (R&D Systems), latent latency-associated peptide (LAP)-TGF-β1 (R&D Systems), β-actin (Sigma), and GAPDH (Cell Signaling).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Hyperactive Transforming Growth Factor-β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

doi: 10.1002/jbmr.1992

Figure Lengend Snippet: Nf1-deficient MSCs exhibit hyperactivation of the Smad pathway and impaired osteoblast differentiation in response to TGF-β1. (A) p-Smad2, Smad2, and GAPDH levels were detected by Western blot in MSCs stimulated with TGF-β1. The quantitative fold change in p-Smad2 was determined relative to the loading control, as shown in the bar graph. The experiment was repeated on three independent occasions with similar results. (B) MSCs were cultured in osteogenic differentiation medium supplemented with TGF-β1. Representative photomicrographs (top panel) show alkaline phosphatase (ALP)-positive osteoblasts (magnification × 200). ALP expression was quantified and normalized to the WT control as shown (bottom panel). n = 3 technical replicates. The assay was repeated twice with similar results using independent cell lines. **p < 0.01, ***p < 0.001. (C) TGF-β1 mRNA expression was measured in Nf1−/− MSCs following retroviral transduction with control vector (MSCV-pac) versus the functional, full-length NF1 GRD construct. n = 3 technical replicates. The experiment was repeated on three independent occasions with similar results. *p < 0.05. (D) p-Smad2, Smad2, and GAPDH levels were detected by Western blot in MSCs stimulated with TGF-β1 following transduction with either MSCV-pac or MSCV-NF1 GRD retroviral vectors. The quantitative fold change in p-Smad2 was determined relative to the level of total Smad2 protein, as shown in the bar graph.

Article Snippet: Immunoblots were performed using antibodies specific to p-Smad2 (Cell Signaling), total Smad2 (Cell Signaling), active TGF-β1 (R&D Systems), latent latency-associated peptide (LAP)-TGF-β1 (R&D Systems), β-actin (Sigma), and GAPDH (Cell Signaling).

Techniques: Western Blot, Cell Culture, Expressing, Transduction, Plasmid Preparation, Functional Assay, Construct

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Hyperactive Transforming Growth Factor-β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

doi: 10.1002/jbmr.1992

Figure Lengend Snippet: TGF-β1 potentiates Nf1 haploinsufficient osteoclast gain-in-functions, which are accompanied by increased activation of the Smad pathway. (A) Osteoclast formation from BMMNCs was induced by M-CSF and RANKL, in the presence or absence of TGF-β1 (1 ng/mL). Representative photomicrographs show multinucleated osteoclasts (magnification ×200) following TRACP staining. Bar graphs represent the mean number of nuclei per osteoclast and the area of TRACP-positive, multinucleated osteoclasts per high power field (HPF), quantified using ImageJ software. n = 4 biological replicates. The experiment was repeated three times with similar results. *p < 0.05, **p < 0.001. (B) Osteoclast formation following TGF-β1 stimulation and increasing doses of SD-208. n = 3 biological replicates. *p < 0.05, **p < 0.01 comparing Nf1+/− versus WT. (C) Osteoclast bone resorption on dentine slices. Representative photomicrographs show resorptive “pits” (magnification × 100). “Pit” area was quantified as shown by the bar graph above. n = 3 biological replicates. ***p < 0.0001. (D) Phosphorylated Smad2 (p-Smad2), total Smad2, and β-actin were measured by Western blot in preosteoclasts stimulated with TGF-β1 (1 ng/mL) in the presence or absence of SD-208 (100 nM). The bar graph shows the fold change in p-Smad2 relative to the loading control. The assay was repeated on two independent occasions with similar results.

Article Snippet: Immunoblots were performed using antibodies specific to p-Smad2 (Cell Signaling), total Smad2 (Cell Signaling), active TGF-β1 (R&D Systems), latent latency-associated peptide (LAP)-TGF-β1 (R&D Systems), β-actin (Sigma), and GAPDH (Cell Signaling).

Techniques: Activation Assay, Staining, Software, Western Blot

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Hyperactive Transforming Growth Factor-β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

doi: 10.1002/jbmr.1992

Figure Lengend Snippet: Hypersecretion of MMP-2/9 promotes excessive latent TGF-β1 activation. (A) Active TGF-β1, latency associated peptide (LAP)-bound TGF-β1, and β-actin were detected by Western blot in protein extracts from the fracture site of Nf1flox/−;Col2.3Cre mice with tibial fracture nonunion and WT controls. The bar graph represents the fold change in the active TGF-β1/LAP-TGF-β1 ratio. n = 5 to 6 mice per group. (B) Activity levels of MMP-2/9 were measured in WT and Nf1+/− myeloid cell conditioned medium by zymography. n = 3 biological replicates. (C) Osteoprogenitors transfected with a Smad luciferase reporter were stimulated with recombinant, active TGF-β1 (1 ng/mL) and latent, LAP-TGF-β1 (100 ng/mL) for 18 hours. n = 3 technical replicates. The experiment was repeated on two independent occasions with similar results. *p < 0.05, **< 0.01. (D) Working model of NF1 skeletal defects mediated by a pathological cycle of increased TGF-β1 synthesis, activation, and Smad signaling.

Article Snippet: Immunoblots were performed using antibodies specific to p-Smad2 (Cell Signaling), total Smad2 (Cell Signaling), active TGF-β1 (R&D Systems), latent latency-associated peptide (LAP)-TGF-β1 (R&D Systems), β-actin (Sigma), and GAPDH (Cell Signaling).

Techniques: Activation Assay, Western Blot, Activity Assay, Zymography, Transfection, Luciferase, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Defective IL-10 signaling in hyper-IgE syndrome results in impaired generation of tolerogenic dendritic cells and induced regulatory T cells

doi: 10.1084/jem.20100799

Figure Lengend Snippet: IL-10 signaling defect in MoDCs leads to the defective generation of tolerogenic DCs and iT reg cells. (A) Representative histograms showing the levels of PD-L1, PD-L2, ILT-3, ILT-4, and ICOS-L produced by untreated immature MoDCs (−) and IL-10–DCs (IL-10) from a control subject and a STAT3 patient are shown at the top. Dashed lines indicate staining with isotype-matched control mAbs. Summary data showing ΔMFI, IL-10–treated minus untreated, of PD-L1, PD-L2, ILT-3, ILT-4, and ICOS-L ( n = 8 each) are at the bottom. (B) Q-PCR analysis of FOXP3 mRNA levels after the co-culture of third-party allogeneic naive CD4 + T cells from a control subject with untreated immature MoDCs (−) or IL-10–DCs (IL-10) from a control subject and a STAT3 patient. Cultures in the absence of naive CD4 + T cells (DC only) and MoDCs (T only) were used as negative controls. Representative data are on the left, and summary data ( n = 8 each) showing fold increase are on the right. (C) Flow cytometric analysis of cytoplasmic FOXP3 protein levels in naive CD4 + T cells co-cultured with untreated immature MoDCs (−) and IL-10–DCs (IL-10) from a control subject and a STAT3 patient. Staining with isotype-matched control mAbs is indicated by dashed lines. Representative data are on the left, and summary data ( n = 8 each) showing percent increase are on the right. (D) CFSE-labeled CD4 + CD25 − responder T cells were cultured alone in the absence (−) or presence of anti-CD3 and anti-CD28 mAbs or with iT reg cells generated by co-culture with control or STAT3 patient immature DCs (iDCs) or IL-10–DCs. After 5 d, the proliferation of CFSE-labeled responder T cells was assessed by flow cytometry. Representative histograms are on the left, and summary data ( n = 8 each) showing the percent increase in nonproliferating cells, numbers in magenta minus numbers in blue, are on the right. (E) Cytokine levels in the supernatants of co-cultures of responder T cells and iT reg cells, as indicated. Representative data are on the left, and summary data ( n = 8 each) showing percent increase are on the right. Data are representative of at least two independent experiments. (F) Q-PCR analysis of FOXP3 mRNA expression after the co-culture of third-party allogeneic naive CD4 + T cells from a control subject with untreated immature MoDCs (−) or IL-10–DCs from a control subject and a STAT3 patient in the absence or presence of exogenous TGF-β1. We show summary data showing relative FOXP3 expression ( n = 8 each) performed in triplicate. Data are representative of at least two independent experiments. (B and E) Graphs show mean ± SD. (A–F) Horizontal bars indicate mean values. **, P < 0.01; ***, P < 0.001.

Article Snippet: We obtained antibodies against ILT-3 (CD85K; 293623), ILT-4 (CD85d; 287219), LAP (latency-associated peptide; TGF-β1; 27235), and GITR (TNFRSF18; 110416) from R&D Systems.

Techniques: Produced, Staining, Co-Culture Assay, Cell Culture, Labeling, Generated, Flow Cytometry, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Defective IL-10 signaling in hyper-IgE syndrome results in impaired generation of tolerogenic dendritic cells and induced regulatory T cells

doi: 10.1084/jem.20100799

Figure Lengend Snippet: PD-L1, ILT-4, and TGF-β1 in response to IL-10–DCs and STAT3 in DCs play a major role in FOXP3 up-regulation. (A–C) Q-PCR analysis of FOXP3 mRNA levels in third-party allogeneic naive CD4 + T cells from control (Cont) subjects co-cultured with untreated immature MoDCs (−) and IL-10–DCs (IL-10) from eight control subjects and eight STAT3 patients. A neutralizing PD-L1 peptide or a control peptide (A), control or ILT-4–neutralizing mAb (B), or control or TGF-β–neutralizing mAb (C) was added where indicated. (D) Q-PCR analysis of FOXP3 mRNA levels in third-party allogeneic naive CD4 + T cells from control subjects and STAT3 patients co-cultured with untreated immature DCs (−) or IL-10–DCs (IL-10) from control subjects and STAT3 patients. Summary data show relative FOXP3 mRNA expression ( n = 8 each) and were performed in triplicate. Data are representative of at least two independent experiments. (A–D) Horizontal bars indicate mean values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: We obtained antibodies against ILT-3 (CD85K; 293623), ILT-4 (CD85d; 287219), LAP (latency-associated peptide; TGF-β1; 27235), and GITR (TNFRSF18; 110416) from R&D Systems.

Techniques: Cell Culture, Expressing