Journal: European journal of immunology

Article Title: TAM receptors attenuate murine NK cell responses via E3 ubiquitin ligase Cbl-b

doi: 10.1002/eji.201948204

Figure Lengend Snippet: LAKs were serum/IL-2-starved for 2 hours, pre-treated with or without Gas6 for 2 hours, and left unstimulated (Unstim) or stimulated with PMA/Iono, anti-NK1.1, or anti-NKG2D for 5 hours and analyzed by flow cytometry using the strategy illustrated in Supporting Information (Figure S1A). (A) Representative flow cytometric plots of IFN-γ+ and CD107a+ LAK cells are shown. (B) The fraction of IFN-γ-expressing and (C) CD107a-expressing LAKs from 4 independent experiments are shown (n = 4 mice for anti-NK1.1 and n=5 mice for anti-NKG2D). (D) Splenoctyes from Poly I:C-treated mice were pre-treated with or without Gas6 for 2 hours and left unstimulated (Unstim) or stimulated with PMA/Iono or anti-NK1.1 for 5 hours and analyzed by flow cytometry using the strategy illustrated in Supporting Information (Figure S1B). Representative flow cytometric plots of IFN-γ+ and CD107a+ NK cells are shown. The fraction of (E) IFN-γ-expressing and (F) CD107a–expressing splenocytes of 4 independent experiments are shown (n =4–5 mice). *p<0.05 and **p<0.01 by paired Student t test, respectively; ns, not significant.

Article Snippet: Recombinant murine Gas6 was purchased from R&D systems (Minneapolis, MN).

Techniques: Flow Cytometry, Expressing

Journal: European journal of immunology

Article Title: TAM receptors attenuate murine NK cell responses via E3 ubiquitin ligase Cbl-b

doi: 10.1002/eji.201948204

Figure Lengend Snippet: (A) LAKs were treated with media (0.05% DMSO) or BMS 777607 (100 nM) for 2 hours in IL-2/serum-free conditions, followed by treatment with or without Gas6 for another 2 hours prior to stimulation with anti-NK1.1 or NKG2D for 5 hours. Data is shown as mean % inhibition {(Untreated - Gas6-treated)/Untreated]*100} ± SEM of 4 independent experiments (n = 4–5 mice for anti-NK1.1 and n=5–6 mice for anti-NKG2D). P values were determined by paired Student t test. (B) WT and Cbl-b KO LAKs were serum/IL-2-starved for 2 hours, followed by Gas6 treatment for 2 hours, and stimulated with anti-NK1.1 or (C) anti-NKG2D antibodies for 24 hours. IFNγ content in the supernatants was measured by ELISA. Data are represented as % of control + SEM of n=4 mice for anti-NK1.1 and n=3 mice for anti-NKG2D. *p< 0.05 and ***p <0.001 by paired Student t test. ns, not significant. (D) LAKs were serum and IL-2-starved for 4 hours followed by Gas6 stimulation for various time points. Cell lysates were immunoblotted with anti-PLCγ1, anti-p85, anti-LAT1, and β-actin antibodies. (E) LAKs from WT and Cbl-b KO mice were serum and IL-2 starved for 4 hours followed by Gas6 stimulation for various time points. Cell lysates were immunoblotted with anti-LAT1 and β-actin antibodies. The relative ratio of LAT1 to β-actin for each time point, normalized to the 0 timepoint for each genotype, is shown below the blots. One representative of three independent experiments is shown.

Article Snippet: Recombinant murine Gas6 was purchased from R&D systems (Minneapolis, MN).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Control

Journal: European journal of immunology

Article Title: TAM receptors attenuate murine NK cell responses via E3 ubiquitin ligase Cbl-b

doi: 10.1002/eji.201948204

Figure Lengend Snippet: (A) HA-tagged Tyro3 was incubated with truncated Cbl-b (TKB+RF), E1, E2, ubiquitin, and ATP in vitro for 1 hour and immunoblotted with anti-HA antibody. (B) Truncated Cbl-b (TKB+RF) was incubated with or without recombinant Tyro3, Axl, or Mer and with or without ATP in vitro and immunoblotted with anti-phosphotyrosine antibody. (C) LAKs were IL-2-starved for 18 hours and serum-starved for 4 hours followed by stimulation with Gas6 for various time points or (D) followed by stimulation with or without Gas6, BMS 777607 with Gas6, or anti-NK1.1 (PK136) with or without Gas6 for 60 minutes. Lysates were de-ubiquitinated with DUB USp2core, followed by immunoprecipitation with anti-phosphotyrosine and immunoblotting with anti-Cbl-b antibodies. (E) 10XHis-tagged Y→F Cbl-b point mutants were incubated with Tyro3 and ATP, followed by immunoblotting with anti-phosphotyrosine antibody. Y3F refers to Y106/133/363F triple mutant. (F) 10XHis-tagged Y→F Cbl-b point mutants were incubated with Tyro3, E1, E2, ubiquitin, and ATP for 1 hour and immunoblotted with anti-ubiquitin antibody. One representative of three independent experiments is shown.

Article Snippet: Recombinant murine Gas6 was purchased from R&D systems (Minneapolis, MN).

Techniques: Incubation, Ubiquitin Proteomics, In Vitro, Recombinant, Immunoprecipitation, Western Blot, Mutagenesis

Journal: European journal of immunology

Article Title: TAM receptors attenuate murine NK cell responses via E3 ubiquitin ligase Cbl-b

doi: 10.1002/eji.201948204

Figure Lengend Snippet: Gas6 binds and activates TAM receptors, which directly phosphorylates Cbl-b. Phosphorylated Cbl-b, in turn, ubiquitinates and targets LAT1 for degradation. NK1.1/NKG2D signaling is attenuated by decreased LAT1 protein, which is required for signal transduction downstream of NK cell activating receptors.

Article Snippet: Recombinant murine Gas6 was purchased from R&D systems (Minneapolis, MN).

Techniques: Transduction

Journal:

Article Title: Role of Gas6 in erythropoiesis and anemia in mice

doi: 10.1172/JCI30375

Figure Lengend Snippet: (A) Western blot analysis of Axl and Mertk in spleen erythroblasts isolated from WT, Tyro3–/–, Axl–/–, and Mertkkd mice. Equal amounts of protein cell lysates were loaded per well. (B) Erythropoiesis of Tyro3–/–, Axl–/– or Mertkkd mice in response to PHZ. Overall genotypic differences versus WT (after Dunnett’s correction for multiple testing) were as follows: Tyro3–/–, P = 0.49 (n = 18); Axl–/–, P = 0.007 (n = 9); Mertkkd, P = 0.97 (n = 5). (C) WT and Axl–/– mice were subjected to PHZ-induced hemolytic anemia (injections of a half dose of PHZ on days 0 and 1) and treated with saline or rGas6 (2 μg daily intraperitoneally). P < 0.05, Axl–/– and Axl–/– plus rGas6 versus WT; P > 0.05, Axl–/– versus Axl–/– plus rGas6.

Article Snippet: Murine rGas6 (986-GS; R&D Systems) and samples were added after dilution in assay buffer.

Techniques: Western Blot, Isolation, Saline

Journal:

Article Title: Role of Gas6 in erythropoiesis and anemia in mice

doi: 10.1172/JCI30375

Figure Lengend Snippet: (A and B) TUNEL staining of spleen 3 days after induction of hemolytic anemia by PHZ. Black staining with TUNEL denotes localized apoptotic cells in WT (A) and Gas6–/– (B) spleen. F, follicle; RP, red pulp. Scale bars: 100 μm. (C) TUNEL assay of WT and Gas6–/– erythroblasts isolated 6 days after PHZ injection. Cells were cultured for 16 h in the presence or absence of recombinant human Epo at the indicated concentrations. The TUNEL assay was quantified by flow cytometry (n = 4); results are expressed as the percentage of apoptotic erythroblasts, cells double-stained for TUNEL and Ter-119. (D) Dead cell count of splenic erythroblasts (Trypan blue+ cells) collected on day 6 after PHZ injection and maintained in culture for 24 h in the presence or absence of Epo (1.5 IU/ml) or Epo plus human rGas6 (400 ng/ml). n = 8 per group. Values are mean ± SEM. *P < 0.05 versus WT.

Article Snippet: Murine rGas6 (986-GS; R&D Systems) and samples were added after dilution in assay buffer.

Techniques: TUNEL Assay, Staining, Isolation, Injection, Cell Culture, Recombinant, Flow Cytometry, Cell Counting

Journal:

Article Title: Role of Gas6 in erythropoiesis and anemia in mice

doi: 10.1172/JCI30375

Figure Lengend Snippet: (A) Adherence of WT versus Gas6–/– erythroblasts to fibronectin (n = 6), a mechanism essential for proliferation, survival, and expansion of these cells. The adhesion defect of Gas6–/– erythroblasts was corrected by the addition of rGas6. (B) Survival response to Epo of WT and Gas6–/– erythroblasts adherent to fibronectin (n = 3). Apoptotic adherent erythroblasts stained by DAPI were counted as cells with nuclear fragmentation. (C) Expression of VLA4 and Ter-119 in WT (n = 9) and Gas6–/– (n = 8) spleen determined by flow cytometry. VLA4 expression is proportional to Ter-119 expression. (D) Adherence of WT versus Gas6–/– erythroblasts to fibronectin in the presence of 0.9% NaCl (vehicle), LY294002, or anti-VLA4 antibody (2 μg/106 cells) (n = 6). Values are mean ± SEM. *P < 0.05. Data were reproduced in 100% C57BL/6 background (not shown).

Article Snippet: Murine rGas6 (986-GS; R&D Systems) and samples were added after dilution in assay buffer.

Techniques: Staining, Expressing, Flow Cytometry

Journal:

Article Title: Role of Gas6 in erythropoiesis and anemia in mice

doi: 10.1172/JCI30375

Figure Lengend Snippet: (A and B) WT (A) and Gas6–/– mice (B) were subjected to PHZ induced hemolytic anemia (injections on day 0 and 1) and treated with saline (control), human rGas6 (2 μg daily intraperitoneally), recombinant human Epo (10 IU every second day intraperitoneally), or a combination of rGas6 and Epo. Overall genotypic differences relative to the control of the same genotype (calculated after Bonferroni correction for multiple testing) were as follows: rGas6-treated WT, P < 0.001; Epo-treated WT, P < 0.001; rGas6-treated Gas6–/–, P = 0.004; Epo-treated Gas6–/–, P = 0.008; rGas6- and Epo-treated Gas6–/–, P < 0.001. (C) Hematocrit levels in transgenic Epo-TAgH mice, a model of stable chronic anemia, treated with rGas6 alone or in combination with Epo at the indicated doses (in μg and IU, respectively). Values are mean ± SEM (n = 6). *P < 0.05 versus control, standard t test.

Article Snippet: Murine rGas6 (986-GS; R&D Systems) and samples were added after dilution in assay buffer.

Techniques: Saline, Control, Recombinant, Transgenic Assay

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: The Journal of Immunology Author Choice

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: Screening anti-murine MerTK Abs using mouse peritoneal macrophages. (A) Flow cytometric analyses of mouse peritoneal macrophages. (B) Kinetic curve of efferocytosis of pHrodo red–labeled apoptotic Jurkat cells by mouse peritoneal macrophages. (C) Single-point pAKT homogeneous time-resolved fluorescence (HTRF) screening of anti-murine MerTK Abs (gray), with positive control Ab AF591 (green) and isotype control (white) using mouse peritoneal macrophages, treated with Gas6-Fc. (D) Two anti-murine MerTK Abs (purple and red) showed dose-dependent inhibitory activities in pAKT HTRF assay, with an anti-MerTK control Ab AF591 (green) and an isotype control (gray). (E) Two anti-murine MerTK Abs (purple and red) showed dose-dependent inhibitory activities of efferocytosis mediated by mouse peritoneal macrophages. pAKT HTRF (D) and efferocytosis (E) curves show representative data (mean ± SD; n = 3) of three independent experiments, respectively.

Article Snippet: Materials Recombinant murine and human MerTK proteins as well as human Gas6-IgG1-Fc fusion protein were made at Genentech.

Techniques: Labeling, Fluorescence, Positive Control, Control, HTRF Assay

Journal: The Journal of Immunology Author Choice

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: Optimization of efferocytosis assay using in vitro differentiated human M2 macrophages. (A) Efferocytosis kinetic curves at different macrophage/apoptotic cell ratios. (B) Comparison of the efferocytosis inhibition curves of anti-MerTK AF891 using regular (gray) versus slow-speed centrifugation (white) to remove cell debris from apoptotic meal; forward/side scatter flow analysis (inlets) showed an 80% decrease of cell debris in apoptotic meal after cleanup. (C) Comparison of the efferocytosis inhibition curves of anti-MerTK AF891 using the default (white) versus optimized (green) image analysis algorithm (IAA). Inlets (original magnification ×10) show the detection of autofluorescence from pHrodo red–labeled cells alone with the default IAA (blue dots, top inlet) and optimized IAA (bottom inlet). (D) Dose-dependent inhibitory activities of anti-MerTK Ab (green) and cytochalasin D (purple) in human M2 macrophage–mediated efferocytosis. (E) Variability of signal across a 96-well plate (top panel) is significantly improved after normalization by macrophage numbers/image (bottom panel); inlets show representative brightfield images (original magnification ×10) of cell seeding variability with cell detection algorithm (yellow outlines). Anti-MerTK AF891 was used to block efferocytosis (gray), and normal goat IgG (from R&D Systems) was used as a negative control (red and green). (F) Comparison of anti-MerTK efferocytosis inhibitory activities of frozen human M2 (blue) versus freshly differentiated M2 macrophages (green). Efferocytosis activity curves show representative data (mean ± SD; n = 4) of at least two independent experiments.

Article Snippet: Materials Recombinant murine and human MerTK proteins as well as human Gas6-IgG1-Fc fusion protein were made at Genentech.

Techniques: In Vitro, Comparison, Inhibition, Centrifugation, Labeling, Blocking Assay, Negative Control, Activity Assay