Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Establishment of a sandwich ELISA for detecting L 59 LAP-DPs. a Schematic diagram of the ELISA for L 59 LAP-DPs. In the ELISA, L 59 LAP-DPs are first captured by coated L59 antibodies (Ab) and then sandwiched by biotin-conjugated anti-LAP antibodies (αLAP-Ab), which form a complex with strep-AP. For detection, an enzyme substrate is added and absorbance at 405 nm was measured. b , c Incubation time- and PLK concentration-dependent increases in the absorbance of the samples containing LAP incubated with PLK. The LAP (final 25 nM) was digested with 12.5 nM (open triangle), 25 nM (open reverse triangle), and 50 nM (open square) PLK, or without PLK (open circle) for 0–90 min, and then the samples were diluted by 1/50 and subjected to the L 59 LAP-DP ELISA ( b ). Also, 25 nM rhLAP was digested with 50 nM PLK (open square) or PLN (cross mark), or PLK in the presence of 5 μM camostat mesilate (open diamond) ( c ). All data were presented as mean ± SD from two different experiments ​*p-value <0.05, **p-value <0.01, ***p-value<0.001 obtained comparing to corresponding control values

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Control

Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Correlation among L 59 LAP-DPs and active TGF-β in the culture medium, and intracellular signal transduction. a , c The ×9CAGA-Luc-transformed CCL64 cells were cultured in PLK-added CM derived from HEK293T cells overexpressing hLTGF-β1. After 6 h, the levels of active TGF-β1 and L 59 LAP-DPs were determined by respective ELISAs ( a ), and the extent of TGF-β signaling was measured by luciferase activity in CCL64 cells ( c ). b , d The scatterplots between the levels of L 59 LAP-DPs and active TGF-β shown in a ( b ) and between increases in L 59 LAP-DP levels and increases in luminescence from the values obtained compared to basal levels in the absence of PLK shown in c ( d ). A significant positive correlation was seen ( b ) * p-value <0.05, ***p-value <0.001 obtained comparing to 0

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Transformation Assay, Cell Culture, Derivative Assay, Luciferase, Activity Assay

Journal: International immunology

Article Title: Recovery from experimental allergic encephalomyelitis is TGF-beta dependent and associated with increases in CD4+LAP+ and CD4+CD25+ T cells.

doi: 10.1093/intimm/dxh390

Figure Lengend Snippet: Fig. 5. TGF-b is involved in recovery from EAE. Frequency of CD4+CD25+LAP+ Tcells (A) and CD4+LAP+ Tcells (B) in naive and EAE-recovered mice. Cells (1.0 3 106) of blood, LN and spleen from naive or EAE-recovered mice (on day 30) were first incubated with anti-CD16/CD32, then stained with 1.0 lg of biotinylated anti-LAP (TGF-b1) polyclonal antibody for 30 min. Cells were washed twice and incubated with PE-conjugated anti-CD25, FITC-conjugated anti-CD4, APC-conjugated streptavidin (1:200 dilution) and 7-AAD. The analysis was performed on a FACScan flow cytometer with CellQuest software. The frequencies of LAP+ cells were determined in the live CD4+CD25+ or CD4+ population (naive mice, n ¼ 12; recovered mice, n ¼ 14, **P < 0.01; ***P < 0.001). (C) Dot plots of CD4+LAP+ T cells in blood and spleen from naive or EAE-recovered mice. (D) Neutralization of TGF-b in vivo. EAE mice were given i.p. 0.4 mg of mouse IgG (open diamonds) or 0.4 mg of anti-TGF-b neutralizing antibody (filled squares) on days 24, 26, 28 and 30 after induction of EAE. Animals were scored daily for symptoms of EAE for 36 days. The result shown here was one of three independent experiments. There were 5–7 mice per group.

Article Snippet: Biotinylated anti-LAP polyclonal antibody was purchased from R&D Systems (catalog no. BAF 246, Minneapolis, MN, USA).

Techniques: Incubation, Staining, Cytometry, Software, Neutralization, In Vivo

Journal: Cancer Research

Article Title: Tumor Cell Invasion Is Promoted by Interstitial Flow-Induced Matrix Priming by Stromal Fibroblasts

doi: 10.1158/0008-5472.can-10-1513

Figure Lengend Snippet: Figure 2. TGF-b1 is necessary for flow-enhanced tumor cell invasion only when fibroblasts are present. Invasion results are normalized to the leftmost condition in each graph unless otherwise noted. A, effects of TGF-b1 neutralization on MDA-MB-435S (TC) invasion (left) or fibroblast (Fb) migration (right) using a function-blocking antibody (a-TGF-b1 block). B, total TGF-b1 levels as measured by ELISA after 18 hours culture with 5 105 tumor cells/mL and 2.5 105 fibroblasts/mL. C, active TGF-b1 levels as measured by TMLC luciferase reporter assay, after 18 hours culture with 2.5 106 reporter cells/mL. The first condition contains only TMLC reporter cells; all other conditions contain the cells noted plus TMLCs. D, left, effects of exogenous TGF-b1 on the invasion of tumor cells alone. Middle, effects of an exogenous TGF-b1 gradient on the invasion of tumor cells alone. Right, effects of exogenous TGF-b1 on tumor cell invasion (in the presence of fibroblasts) and fibroblast invasion alone. Tumor cell and fibroblast invasion are normalized to their respective flow, no TGF-b1 condition. *, P < 0.05 versus matching static condition; #, P < 0.05 between indicated groups.

Article Snippet: As appropriate, 0.45 mg/mL heparan sulfate, 0.45 mg/mL chondroitin sulfate A, 0.3 U/mL heparinase III, 100 mmol/L blebbistatin, 10 mmol/L ML-7 (Sigma-Aldrich), 1 mg/mL mouse anti-human TGF-b1 antibody or mouse IgG1 isotype control (R&D Systems), rhTGF-b1 (PeproTech), 10 mmol/L GM6001 (Millipore), and 2 mg/mL cell-permeable C3 transferase (Cytoskeleton Inc.) were added to the matrix and medium.

Techniques: Neutralization, Migration, Blocking Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay

Journal: Cancer Research

Article Title: Tumor Cell Invasion Is Promoted by Interstitial Flow-Induced Matrix Priming by Stromal Fibroblasts

doi: 10.1158/0008-5472.can-10-1513

Figure Lengend Snippet: Figure 6. Hypothesized mechanism of flow- and fibroblast-enhanced tumor cell migration. Stromal fibroblasts secrete latent TGF-b1 that binds ECM. Interstitial flow enhances activation and availability of TGF-b1, stimulates collagen degradation, and increases fibroblast migration. As the fibroblast moves, it primes the ECM through Rho-dependent contractility. Nearby tumor cells (already invading by CCR7-dependent autologous chemotaxis) take advantage of the primed matrix to enhance their invasion.

Article Snippet: As appropriate, 0.45 mg/mL heparan sulfate, 0.45 mg/mL chondroitin sulfate A, 0.3 U/mL heparinase III, 100 mmol/L blebbistatin, 10 mmol/L ML-7 (Sigma-Aldrich), 1 mg/mL mouse anti-human TGF-b1 antibody or mouse IgG1 isotype control (R&D Systems), rhTGF-b1 (PeproTech), 10 mmol/L GM6001 (Millipore), and 2 mg/mL cell-permeable C3 transferase (Cytoskeleton Inc.) were added to the matrix and medium.

Techniques: Migration, Activation Assay, Chemotaxis Assay

Journal: bioRxiv

Article Title: Major histocompatibility complex class II-expressing bone marrow megakaryocytes activate CD4 + T cells and induce regulatory T cell fate

doi: 10.1101/2025.11.21.689743

Figure Lengend Snippet: (A) Representative images (left) and quantification of MK numbers and MK area (right) in MHC KO and Cre ( - ) mice; n =10 mice/ genotype. (B) Colocalization analysis of LAP and CD41 on femoral cryosections of MHC KO and Cre ( - ) mice; n =4 mice/ genotype. (C) Total MK counts 24 hours after LPS or Poly (I:C) administration; n= 5 mice/ genotype. (D) Frequency of CD40, CD53, CD80, CD83, CD86 (+) MKs 24 hours after LPS or Poly (I:C) administration; n = 5 mice/ genotype. Data are shown as mean ± SD; graphs represent data from at least 3 independent experiments. Statistics performed with unpaired 2-tailed Student t test (A–C) and 1-way ANOVA with Tukey’ multiple comparisons test at 95.00% CI (C).

Article Snippet: MKs and platelets were stained overnight using antibodies against LAP (AF246-NA, R&D Systems), a-tubulin (322588, ThermoFisher Scientific), proplatelet basic protein (PPBP), (PA5-47947, Invitrogen), CD42a (M-051-0, Emfret Analytics), Ovalbumin, Alexa FluorTM 647 Conjugate (Thermo Fisher Scientific), nuclei were stained using DAPI.

Techniques: