murine gas6  (R&D Systems)

Journal: Theranostics

Article Title: Regulatory T-cells regulate neonatal heart regeneration by potentiating cardiomyocyte proliferation in a paracrine manner

doi: 10.7150/thno.32734

Figure Lengend Snippet: The most significantly upregulated genes expressed by heart Treg during neonatal heart regeneration. Foxp3 + Treg are purified from the spleen or heart at day 7 post CI to P3 ICR mice. C1: upregulated genes in splenic naïve Treg; C2: upregulated genes in Treg following activation by neoantigens released during cryoinfarction in the heart.

Article Snippet: After that, they were cocultured with in vitro stimulated Treg in a ratio of cardiomyocytes: Treg as 3:1, Treg supernatant-containing dark medium (1:1) or 50 ng/ml murine CCL24 (Biolegend, 585102), 100 ng/ml murine GAS6 (RnD systems, 8310-GS-050), 1 ug/ml murine GRN (Lifespan biosciences, LS-G3786-10) or 100 ng/ml murine amphiregulin (RnD systems, 989-AR-100) at 37°C for 1 day before analysis.

Techniques: Purification, Activation Assay, Immunopeptidomics

Journal: Theranostics

Article Title: Regulatory T-cells regulate neonatal heart regeneration by potentiating cardiomyocyte proliferation in a paracrine manner

doi: 10.7150/thno.32734

Figure Lengend Snippet: Treg directly promote proliferation of mouse neonatal cardiomyocytes in a paracrine manner. Immunocytochemistry for cTnT + (red) and Ki67 + (green), pH3 + (green) or Aurora B + (green) cells at day 1 after coculture of (A) CD3 + CD4 + hCD2 + Treg, Treg supernatant (SN), or (G) the combination of CCL24, GAS6 and AREG (Pool 3) with mouse neonatal cardiomyocytes of P1 ICR hearts, scale bars: 50 um. Quantification of (B) the absolute number of total cTnT + cardiomyocytes after cocultured for 3 days; or (C) %Ki67 + cTnT + , (D) %pH3 + cTnT + or (E) %Aurora B + cTnT + proliferating cardiomyocytes among total cTnT + cardiomyocytes based on (A). Quantification of proliferating cardiomyocytes after cultured with (F) the respective paracrine factors or (H-J) Pool 3 for 1 day. Data are presented as mean±S.D., n = 3 independent experiments, *P<0.05, **P<0.01.

Article Snippet: After that, they were cocultured with in vitro stimulated Treg in a ratio of cardiomyocytes: Treg as 3:1, Treg supernatant-containing dark medium (1:1) or 50 ng/ml murine CCL24 (Biolegend, 585102), 100 ng/ml murine GAS6 (RnD systems, 8310-GS-050), 1 ug/ml murine GRN (Lifespan biosciences, LS-G3786-10) or 100 ng/ml murine amphiregulin (RnD systems, 989-AR-100) at 37°C for 1 day before analysis.

Techniques: Immunocytochemistry, Cell Culture

Journal: Nature

Article Title: The E3 Ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells

doi: 10.1038/nature12998

Figure Lengend Snippet: a, In vitro Cbl-b-dependent ubiquitylation of Tyro3, Axl, and Mer (anti-Ub). Control, no TAM receptors. Loading controls are shown. b, IFN-γ+ Cbl-b+/+ and Cbl-b-/- NK cells (%) stimulated with anti-NKG2D Abs in the presence of soluble Gas6. *P<0.05 (One-way ANOVA, Dunnett’s post hoc test, n=6/5). c, TAM receptors surface expression in NK cells after stimulation with Gas6. *P<0.05 (two-way ANOVA, Bonferroni’s post hoc test, n=10-13). d, Chemical structure of the TAM receptor kinase inhibitor LDC1267. e, IC50 values for the indicated protein kinases as determined by tracer assays. f, Remaining activity (compared to DMSO control) in 456 kinases treated with 1µM LDC1267 g, IFN-γ+ Cbl-b+/+ and Cbl-b-/- NK cells (%) pre-treated with vehicle or LDC1267 and then stimulated with anti-NKG2D Abs and Gas6. *P<0.05, n.s., not significant (Student’s t-test, n=11/8.). h, In vivo NK cytotoxicity towards RMA-Rae1 cells in mice treated with vehicle or LDC1267. *P<0.05, **P<0.01 (One-way ANOVA, Tukey’s post hoc test, n=16/14/9/10). i, Tumor-to-lung areas in B16F10 tumor-bearing B6 mice untreated or adoptively transplanted with NK cells ex vivo treated with vehicle or LDC1267. *P<0.05 and **P<0.01 (One-way ANOVA, Tukey’s post hoc test, n=16/16/16/10/10). Data in b,c,g-i are mean ± s.e.m.

Article Snippet: For Gas6 and Cbl-b co-immunoprecipitations, HeLa cells were serum-starved overnight and stimulated at 37°C with 450ng/ml recombinant His-tagged murine Gas6 (R&D System) for different time periods, and then lysed as described above.

Techniques: In Vitro, Expressing, Activity Assay, In Vivo, Ex Vivo