Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet: Endothelial, fibroblast and cardiomyocyte cell purification from mouse hearts (A) Scheme on the experimental procedures to isolate the indicated cell types from mouse hearts. (B) Heatmap of cell marker gene expression in RNA from isolated cardiomyocytes (CM), endothelial cells (EC) or fibroblasts (FB) after sham or the indicated time point after TAC surgery. (C) Cardiac endothelial cells and fibroblasts were isolated from mouse hearts and stained for the endothelial markers CD31 and CD102, for the leukocyte marker CD45, and the fibroblast marker Mefsk4. Subsequently, flow cytometric analyses were performed and representative results are shown here. The numbers indicated in each quadrant indicates the percentage of cells localized in that particular quadrant. (D) RNA from the different cell types after sham or 1 and 8 weeks after TAC was subjected to RNA sequencing. The differences in overall gene expression patterns were visualized by a principal component analysis.

Article Snippet: CD31 Antibody, anti-mouse , Miltenyi Biotec , Clone 390.

Techniques: Purification, Marker, Gene Expression, Isolation, Staining, RNA Sequencing

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet:

Article Snippet: CD31 Antibody, anti-mouse , Miltenyi Biotec , Clone 390.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Magnetic Beads, Recombinant, Clinical Proteomics, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cancer Science

Article Title: Synthetic disulfide-bridged cyclic peptides mimic the anti-angiogenic actions of chondromodulin-I

doi: 10.1111/j.1349-7006.2012.02276.x

Figure Lengend Snippet: Effects of the synthetic and tailed chondromodulin-I (ChM-I) cyclic peptide on tumor angiogenesis and growth in an animal model in which OUMS-27 cells (5 × 10 6 cells) were subcutaneously inoculated in the backs of 4 week-old nude mice. (A) Time-course of tumor volume changes. When the tumor volume reached about 45 mm 3 , each mouse was injected around the tumor mass each day for the initial 5 days with PBS (50 μL) alone (♢), PBS containing 4.3 nmol (20 μg) ChM-I cyclic peptide with a tail (○), or PBS containing 0.2 nmol (5 μg) recombinant human ChM-I (rhChM-I) (●) (arrows). The tumor volumes were determined by the width 2 × length × 0.52. Values are the means ± SD from at least six animals per group. (B) Gross appearance of tumors excised on day 21. Bar, 10 mm. (C) Immunohistochemical staining of the CD31-positive vasculature. On day 21, tumor tissues were excised, fixed, and cross-sectioned. The sections were then stained with toluidine blue (left panels), and semi-serial sections were stained with an anti-type II collagen antibody ( green signal in the right panels) and an anti-CD31 antibody ( red signal in the right panels), respectively. Bars, 100 μm. (D) The CD31-positive area was measured as described in the methods section. Values are the means ± SD of five tumors per group. * P < 0.05, ** P < 0.01.

Article Snippet: Anti-CD31 antibody and anti-collagen type II antibody were obtained from BD PharMingen (San Diego, CA, USA) and Rockland (Gilvertsville, PA, USA), respectively.

Techniques: Animal Model, Injection, Recombinant, Immunohistochemical staining, Staining

Journal: Journal of Biological Chemistry

Article Title: T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component

doi: 10.1074/jbc.m110.201236

Figure Lengend Snippet: FIGURE 1. Quantitative analysis of TCR-dependent protein tyrosine phosphorylation. A, experimental workflow is shown. Jurkat cells were metaboli- cally labeled with three combinations of arginine and lysine containing light (R0, K0) or heavy (R6, K4 or R10, K8) isotopes until complete incorporation (as de- scribed under “Experimental Procedures”). Equal numbers of cells for each labeling condition were divided into two series (1 and 2), activated with anti-CD3 Ab at 37 °C for the indicated times, and immediately lysed in ice-cold lysis buffer. Post-nuclear lysates of each activation series were mixed in a 1:1:1 ratio and subjected to Tyr(P) (pY) immunoprecipitation for 60–90 min at 4 °C using a mixture of three anti-Tyr(P) Abs coupled to beads (4G10, Tyr(P)-99, Tyr(P)- 20). Beads were washed and eluted with phenyl phosphate, and eluates were separated by SDS-PAGE. 10 slices per gel lane were cut and digested with trypsin overnight, and the resulting tryptic peptides were analyzed by LC-MS/MS. After protein identification and quantitation, relative protein abundance in each activation series were bridged and normalized using quantitation of the common time point (0.5 min), thus, resulting in a continuous activation profile over 5 time points. B, 758 proteins were confidently identified from three independent SILAC experiments. 141 proteins that showed a significant increase (1.6-fold) above basal levels in anti-Tyr(P) immunoisolates after TCR stimulation were selected, of which 77 were retained after further manual validation. Numbers within bars indicate tyrosine-phosphorylated proteins identified in each set. C, over-represented domains within identified protein se- quences from the three SILAC experiments; most domains are involved in signaling events. ArfGAP, GTPase activating proteins toward Arf; CH, calponin ho- mology domain; RRM, RNA recognition motif; C2, calcium-dependent phospholipid binding domain; RhoGAP, Rho GTPase activating protein domain; Rho- GEF, guanine nucleotide exchange factor for Rho GTPases domain; PH, Pleckstrin homology domain; SH2, Src homology domain 2; SH3, Src homology domain 3. D, analysis of activated signaling pathways by cross-correlation with the signaling pathways data base Kegg is shown.

Article Snippet: Mouse monoclonal antibodies (Abs) used included: antiphosphotyrosine (Tyr(P)) (4G10, Millipore, PY99, Santa Cruz Biotechnology, and PY20, BD Transduction Laboratories); anti-LCK (lymphocyte-specific kinase; 3A5, Santa Cruz); antiZAP-70 (2F3.2), anti-LAT (2E9), anti-phospho-LAT (Tyr226) (Millipore); anti-CD3 (Tyr142, BD Biosciences); anti-phospho-ERK1/2 (E10, Cell Signaling Technology); anti-GAPDH (6C5, Calbiochem); anti-One-STrEP-Tag mAb (StrepMAB Classic, IBA bioTAGnology); anti-human CD3 (UCHT-1) and anti-SLP-76 (SLP-76/03, AbD Serotec); anti-human CD28 (CD28.2, BioLegend).

Techniques: Phospho-proteomics, Labeling, Lysis, Activation Assay, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Quantitative Proteomics, Multiplex sample analysis, Biomarker Discovery, Binding Assay, Protein-Protein interactions

Journal: Journal of Biological Chemistry

Article Title: T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component

doi: 10.1074/jbc.m110.201236

Figure Lengend Snippet: FIGURE 4. Themis is a new component of the SLP-76LAT signalosome. A, shown is a THEMIS-One STrEP tag (OST) pulldown assay using Streptactin-Sep- harose after anti-CD3 stimulation in Jurkat cells transfected with non-targeting control shRNA or a shRNA construct targeting LCK (70% knockdown effi- ciency). IB, immunoblot; pY, Tyr(P). B, THEMIS IP from anti-CD3 stimulated LAT-deficient (J.CaM2.5) and reconstituted cells is shown. C, THEMIS IP from anti- CD3 stimulated SLP-76-deficient (J14) and reconstituted cells is shown. D, THEMIS IPs from resting or anti-CD3-stimulated Jurkat cells is shown. Specific THEMIS antibody saturated with the peptide against which the antibody was raised served as a control. E, a THEMIS-OST pulldown assay from resting or CD3-stimulated Jurkat cells stably expressing THEMIS-OST using biotin-saturated Streptactin-Sepharose as control is shown. Both sets of blots were probed with antibodies against TCR-proximal signaling proteins.

Article Snippet: Mouse monoclonal antibodies (Abs) used included: antiphosphotyrosine (Tyr(P)) (4G10, Millipore, PY99, Santa Cruz Biotechnology, and PY20, BD Transduction Laboratories); anti-LCK (lymphocyte-specific kinase; 3A5, Santa Cruz); antiZAP-70 (2F3.2), anti-LAT (2E9), anti-phospho-LAT (Tyr226) (Millipore); anti-CD3 (Tyr142, BD Biosciences); anti-phospho-ERK1/2 (E10, Cell Signaling Technology); anti-GAPDH (6C5, Calbiochem); anti-One-STrEP-Tag mAb (StrepMAB Classic, IBA bioTAGnology); anti-human CD3 (UCHT-1) and anti-SLP-76 (SLP-76/03, AbD Serotec); anti-human CD28 (CD28.2, BioLegend).

Techniques: Strep-tag, Transfection, Control, shRNA, Construct, Knockdown, Western Blot, Stable Transfection, Expressing

Journal: Journal of Biological Chemistry

Article Title: T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component

doi: 10.1074/jbc.m110.201236

Figure Lengend Snippet: FIGURE 5. THEMIS is a positive regulator of TCR-induced signaling. A, IL-2 ELISAs of shControl, shTHEMIS1, shTHEMIS2, and shLAT Jurkat cells stimulated with plate-bound anti-CD3 and soluble anti-CD28 (left panel) or staphylococcal enterotoxin E-pulsed Raji B cells (right panel) for 24 h is shown. Immunoblot (IB) analysis of the cell lines used (lower panel, shTHEMIS1 (60% knockdown), shTHEMIS2 (90% knockdown), shLAT (60% knockdown), and the non-targeting shControl). B, IL-2-luciferase assay of shControl and shTHEMIS2 (90% knockdown) is shown. IL-2-luciferase cells were transduced with either empty vector or an shRNA-resistant mutant of THEMIS (right panel) and stimulated with staphylococcal enterotoxin E-pulsed Raji B cells (left panel). C, TCR stimulation-in- duced IL-2 secretion in peripheral CD4CD25 T cells from wild-type and Themis knock-out mice is shown. Conventional CD4 cells were purified by nega- tive selection and stimulated with plate-bound anti-CD3 and soluble anti-CD28 Ab for 48 h. IL-2 concentrations in supernatants were measured by ELISA. Shown are data from three mice of each group, p 0.03.

Article Snippet: Mouse monoclonal antibodies (Abs) used included: antiphosphotyrosine (Tyr(P)) (4G10, Millipore, PY99, Santa Cruz Biotechnology, and PY20, BD Transduction Laboratories); anti-LCK (lymphocyte-specific kinase; 3A5, Santa Cruz); antiZAP-70 (2F3.2), anti-LAT (2E9), anti-phospho-LAT (Tyr226) (Millipore); anti-CD3 (Tyr142, BD Biosciences); anti-phospho-ERK1/2 (E10, Cell Signaling Technology); anti-GAPDH (6C5, Calbiochem); anti-One-STrEP-Tag mAb (StrepMAB Classic, IBA bioTAGnology); anti-human CD3 (UCHT-1) and anti-SLP-76 (SLP-76/03, AbD Serotec); anti-human CD28 (CD28.2, BioLegend).

Techniques: Western Blot, Knockdown, Luciferase, Transduction, Plasmid Preparation, shRNA, Mutagenesis, Knock-Out, Purification, Selection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: T Cell Receptor (TCR)-induced Tyrosine Phosphorylation Dynamics Identifies THEMIS as a New TCR Signalosome Component

doi: 10.1074/jbc.m110.201236

Figure Lengend Snippet: FIGURE 6. THEMIS positively modulates NFAT/AP-1 and ERK activity. A, shown is an NFAT/AP-1-luciferase assay of shControl, shTHEMIS1 (60% knockdown), and shTHEMIS2 (90% knockdown) NFAT/AP-1-luciferase Jurkat cells after stimulation with plate-bound anti-CD3 and soluble anti-CD28 (left panel) and immunoblot (IB) analysis of THEMIS expression in the cell lines used (right panel). RLU, relative light units. B, an NF-B-luciferase assay of shControl, shTHEMIS1, and shTHEMIS2 Jurkat cells stimulated as in A (for THEMIS expression levels in cell lines used see Fig. 5A, lower panel). C, im- munoblots of ERK1/2 phosphorylation kinetics in shControl and shTHEMIS2 (90% knockdown) Jurkat cells in three independent experiments using de- creasing anti-CD3 concentrations (total ERK1/2 and ZAP-70 blots are shown as loading controls) are shown.

Article Snippet: Mouse monoclonal antibodies (Abs) used included: antiphosphotyrosine (Tyr(P)) (4G10, Millipore, PY99, Santa Cruz Biotechnology, and PY20, BD Transduction Laboratories); anti-LCK (lymphocyte-specific kinase; 3A5, Santa Cruz); antiZAP-70 (2F3.2), anti-LAT (2E9), anti-phospho-LAT (Tyr226) (Millipore); anti-CD3 (Tyr142, BD Biosciences); anti-phospho-ERK1/2 (E10, Cell Signaling Technology); anti-GAPDH (6C5, Calbiochem); anti-One-STrEP-Tag mAb (StrepMAB Classic, IBA bioTAGnology); anti-human CD3 (UCHT-1) and anti-SLP-76 (SLP-76/03, AbD Serotec); anti-human CD28 (CD28.2, BioLegend).

Techniques: Activity Assay, Luciferase, Knockdown, Western Blot, Expressing, Phospho-proteomics