murine tsp 1  (R&D Systems)

Journal: PLoS ONE

Article Title: In Vitro and In Vivo Evidence that Thrombospondin-1 (TSP-1) Contributes to Stirring- and Shear-Dependent Activation of Platelet-Derived TGF-β1

doi: 10.1371/journal.pone.0006608

Figure Lengend Snippet: (A) Immunoblots of WT and Thbs1 −/− mice sera demonstrate absence of TSP-1 in the Thbs1 −/− mice. (B, C, D) Sera from WT (n = 23) and Thbs1 −/− (n = 23) mice were stirred (S) at 1,200 rpm or left unstirred (US) for 120 min at 37°C and then total (B) and active (C, D) TGF-β1 were measured; the latter was expressed either as an absolute value (ng/mL) (C) or as a percentage of total TGF-β1 (D). Levels of active TGF-β1 increased less in Thbs1 −/− than WT mice with stirring [p = 0.057 (absolute values) and p = 0.016 (percentages of total TGF-β1) for interaction by ANOVA]. The post-stirring values were also higher in WT than Thbs1 −/− mice [p = 0.19 (absolute values) and p = 0.001 (percentages of total TGF-β1) by t-test]. (E, F) Sera from WT (n = 10) or Thbs1 −/− (n = 10) mice were either incubated at 37°C (−) or subjected to shear (+) at 1,800 s −1 at 37°C for 120 min. Active TGF-β1 increased more in WT mice, both in terms of absolute values (p = 0.18 by t-test) (E) and as percentages of total TGF-β1 (p = 0.039 by t-test) (F).

Article Snippet: A murine monoclonal antibody (mAb) to TSP-1 (Ab-11) was obtained from NeoMarkers (Fremont, CA), a chicken polyclonal IgY antibody to human TGF-β1 and recombinant human TSP-1 (rhTSP-1) were obtained from R&D Systems (Minneapolis, MN), TSP-1 inhibitor and control peptides were obtained from Ana Spec Inc. (San Jose, CA), and Streptavidin coupled to magnetic beads was obtained from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Incubation

Journal: PLoS ONE

Article Title: In Vitro and In Vivo Evidence that Thrombospondin-1 (TSP-1) Contributes to Stirring- and Shear-Dependent Activation of Platelet-Derived TGF-β1

doi: 10.1371/journal.pone.0006608

Figure Lengend Snippet: (A) Thrombin-induced aggregation of washed platelets from WT and Thbs1 −/− mice. (B, C, D) Platelet releasates were either left unstirred (US) at 37°C or stirred (S) at 1,200 rpm for 120 min and then total (B) and active (C, D) TGF-β1 were measured. (B) The decline of total TGF-β1 in stirred versus unstirred platelet releasates was similar in WT and Thbs1 −/− mice. (C, D) Active TGF-β1 increased more in WT than Thbs1 −/− mice [p = 0.004 (absolute values) and p = 0.005 (percentages of total TGF-β1) for interaction by ANOVA]. The final values were also higher in WT vs Thbs1 −/− mice (p = 0.005 and p = 0.008 by t-test). (E) Immunoblotting of platelet releasates obtained from WT or TSP-1 null mice demonstrated absence of TSP-1 protein in a Thbs1 −/− mouse (lane 4); addition of 20 µg/mL of rhTSP-1 protein to the Thbs1 −/− mouse sample (lane 2) achieved a TSP-1 level similar to those in a WT mouse sample (lane 1) and a human platelet releasate (lane 3). (F) Adding rhTSP-1 (+) versus BSA (−) corrected the defect in stirring-induced activation of TGF-β1 in Thbs1 −/− mice.

Article Snippet: A murine monoclonal antibody (mAb) to TSP-1 (Ab-11) was obtained from NeoMarkers (Fremont, CA), a chicken polyclonal IgY antibody to human TGF-β1 and recombinant human TSP-1 (rhTSP-1) were obtained from R&D Systems (Minneapolis, MN), TSP-1 inhibitor and control peptides were obtained from Ana Spec Inc. (San Jose, CA), and Streptavidin coupled to magnetic beads was obtained from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: In Vitro and In Vivo Evidence that Thrombospondin-1 (TSP-1) Contributes to Stirring- and Shear-Dependent Activation of Platelet-Derived TGF-β1

doi: 10.1371/journal.pone.0006608

Figure Lengend Snippet: (A) The proteins in human platelet releasates were labeled with MPB (100 µM) for 30 min either before (−) or after (+) shear for 2 hours. The labeled proteins were either analyzed directly (left two lanes) or after affinity-purification using Streptavidin-coupled beads (right two lanes). Shearing led to a dramatic decrease in intensity of the HRP reaction in select regions. (B) One of the MPB-labeled proteins (boxed) that was most affected by shearing was identified as TSP-1 by LC-MS/MS analysis. (C) Platelet releasates were passed through either a control-Sepharose column (Con) or a thiol-Sepharose column (Thiol) and then labeled with MPB. Depletion of thiol-reactive proteins by the column was analyzed by reaction of the separated proteins with Streptavidin (left panel) and depletion of TSP-1 protein was measured by immunoblotting with an anti-TSP-1 antibody (right panel). Nearly all of the proteins that labeled with MPB from the control column were not labeled after passage through the thiol-Sepharose column. (D) Effect of increasing time of exposure to shear on depletion of TSP-1 from platelet releasates. MPB labeling of TSP-1 was concordantly reduced with the loss to TSP-1 protein during shear as judged by reaction with Streptavidin-HRP (left panel) and immunoblotting with an anti-TSP-1 antibody (middle panel). TGF-β1 depletion was much less pronounced as judged by immunoblotting with an anti-TGF-β1 antibody (right panel). (E) Addition of MPB (100 µM) before shear partially prevented the loss of TSP-1 protein as shown by immunoblotting with an anti-TSP-1 antibody. Addition of the other thiol-reactive reagents, BMCC (F) or NEM (G), similarly protected against loss of TSP-1. Vertical lines in (F) indicate deletion of intermediate lanes from the same gel.

Article Snippet: A murine monoclonal antibody (mAb) to TSP-1 (Ab-11) was obtained from NeoMarkers (Fremont, CA), a chicken polyclonal IgY antibody to human TGF-β1 and recombinant human TSP-1 (rhTSP-1) were obtained from R&D Systems (Minneapolis, MN), TSP-1 inhibitor and control peptides were obtained from Ana Spec Inc. (San Jose, CA), and Streptavidin coupled to magnetic beads was obtained from Invitrogen (Carlsbad, CA).

Techniques: Labeling, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: iScience

Article Title: CD47 restricts antiviral function of alveolar macrophages during influenza virus infection

doi: 10.1016/j.isci.2022.105540

Figure Lengend Snippet: CD47 is involved in the regulation of hemoglobin expression in alveolar macrophages Mice were i.n. infected with IAV. aMФ were isolated from WT and CD47 −/− mice 3 dpi. (A) Transcriptome analysis of isolated WT and CD47 −/− aMФ were assessed by an Affymetrix MicroArray. Hemoglobin alpha (HBA) (B) and beta (HBB) (C) chains were detected by flow cytometry analysis in aMФ isolated from WT and CD47 −/− mice. Representative histograms for HBA and HBB are shown. Mean fluorescence intensity (MFI) for HBA and HBB from aMФ are shown (n = 4). (D) Whole hemoglobin (HB) concentration was assessed in BALF at indicated time points by ELISA (n = 7–17). (E) aMФ were isolated from infected WT and CD47 −/− mice 3 dpi and stimulated ex vivo with thrombospondin-1 (TSP-1). After 24 h, HB concentration in the supernatants was measured by ELISA (n = 4). (F) Type II alveolar epithelial cells (AEC II) were isolated from the lung of naive WT mice and incubated with 1 μg/mL Alexa Fluor 647 (AF647)-labeled HB for 30 min. Representative flow cytometry plots are shown. F) Percentages of HB + AEC II cells are shown (n = 4). (G) Isolated cells from lungs of WT mice were incubated with 1 μg/mL AF647-labelled HB for 30 min. Percentages of HB + aMФ, iMФ, and DCs are shown. Results depicted were obtained from one (A) or two independent experiments (C, D, E, G, and H). Data shown are mean ± SEM. One-way ANOVA with Tukey’s multiple-comparisons post-test (D and E) or Student’s t test (F) were performed. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗∗ = p < 0.0001.

Article Snippet: Cells were incubated with 1 μg/mL and 5 μg/mL murine TSP-1 (R&D) in 300 μL Medium on a 24 well plate for 24 h. Afterward, supernatants were collected and analyzed for HB concentration by ELISA.

Techniques: Expressing, Infection, Isolation, Microarray, Flow Cytometry, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Ex Vivo, Incubation, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Thrombospondin-1 Is a Transcriptional Repression Target of PRMT6 *

doi: 10.1074/jbc.M109.005322

Figure Lengend Snippet: Interference with the activity of TSP-1 restores cell migration properties to PRMT6-deficient U2OS cells. A, cell lysates or the conditioned media from control and PRMT6 siRNA-treated U2OS cells were immunoblotted with α-PRMT6, α-TSP-1, and α-tubulin as a loading control. The molecular mass markers are shown on the left in kDa. B, cell migration of siGFP- and siPRMT6-treated U2OS cells was assayed in the presence of either 20 μm neutralizing TSP-1 (GGWSHW) or the control (GGYSHW) peptide in the conditioned media. The data represent the means ± S.D. of two independent experiments performed in triplicate. Statistically significant differences, as compared with respective control conditions, are indicated by **, p < 0.01 (Student's t test). C, cell migration assays were also performed with the presence of either 10 μg/ml blocking α-TSP-1 or immunoglobulin G in the conditioned media. The data represent the means ± S.D. of two independent experiments performed in triplicate. Statistically significant differences, as compared with respective control conditions, are indicated by **, p < 0.01 (Student's t test).

Article Snippet: TSP-1 Promoter Activity Assays The pMTSP-1 vector containing −2,800 to +48 bp of the murine TSP-1 promoter upstream of firefly luciferase reporter gene has been described ( 49 ) and was kindly provided by Dr. Paul Bornstein (University of Washington, Seattle) via Addgene (Addgene plasmid 12409; Cambridge, MA).

Techniques: Activity Assay, Migration, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Thrombospondin-1 Is a Transcriptional Repression Target of PRMT6 *

doi: 10.1074/jbc.M109.005322

Figure Lengend Snippet: PRMT6 regulates the activity of the TSP-1 promoter by methylating H3R2 in U2OS cells. A, activity of the TSP-1 promoter luciferase (Luc.) reporter gene was assessed in U2OS cells transfected with either siGFP or siPRMT6. The luciferase activity was normalized to Renilla, and the latter was used to assess transfection efficiency. The transfected cells were lysed and assayed for luciferase activity as described under “Experimental Procedures.” The results are expressed as a ratio of firefly luciferase (FfLuc) on Renilla luciferase (RenLuc) fold induction activities. Data represent the means ± S.D. of two independent experiments performed in triplicate. Statistically significant differences as compared with control conditions are as follows: *, p < 0.05; ***, p < 0.001 (Student's t test). B, activity of the TSP-1 promoter luciferase reporter gene was assessed in U2OS cells transfected with either empty vector (pVAX1), wild-type PRMT6 (pVAX1 PRMT6 WT), and catalytically inactive PRMT6 (pVAX1 PRMT6 VLD:KLA). The activity of a TSP-1 promoter luciferase reporter gene was assessed as in A. C, chromatin immunoprecipitation assays were performed with control IgG and α-PRMT6 antibodies. DNA oligonucleotides from the TSP-1 promoter were used in a PCR assay to amplify a DNA fragment. The latter was visualized by ethidium bromide-stained agarose gel electrophoresis. D, analysis of the occupancy of H3R2me2a and H3K4me3 histone marks at the TSP-1 gene promoter by ChIP analysis. ChIP assays were performed with chromatin prepared from U2OS cells transfected or not with control siGFP or siPRMT6. After 72 h, the cells were harvested for ChIP analysis using the indicated antibodies and a primer pair located in the promoter region of TSP-1. ChIPs experiments were performed in duplicate, and the results shown are representative of the duplicates.

Article Snippet: TSP-1 Promoter Activity Assays The pMTSP-1 vector containing −2,800 to +48 bp of the murine TSP-1 promoter upstream of firefly luciferase reporter gene has been described ( 49 ) and was kindly provided by Dr. Paul Bornstein (University of Washington, Seattle) via Addgene (Addgene plasmid 12409; Cambridge, MA).

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Staining, Agarose Gel Electrophoresis, ChIP-chip