Journal: International journal of molecular sciences

Article Title: Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

doi: 10.3390/ijms25126630

Figure Lengend Snippet: Figure 2. Gas6 and Protein S bioavailabilities peak at different times of the light–dark cycle. Analysis of the mRNA (A,C) and protein (B,D) expression profiles for Gas6 (A,B) and Protein S (C,D) in the RPE/choroid, retina, or IPM for wildtype (wt, blue) and β5−/−mice (β5 ko, pink) at different times of day as indicated. (A) qPCR experiments allowed us to show that Gas6 mRNA expression levels are slightly increased just before (retina) and after (RPE/choroid) the phagocytic peak in wt mice. Gas6 expression levels were lower in the RPE/choroid of β5−/−mice between peak phagocytosis time and 22.00 while levels were unchanged in the retina fraction. (B) Corresponding protein quantification and representative immunoblots in the IPM of fellow eyes showed a decrease at light onset followed by a marked increase at the time of the phagocytic peak in wt animals. In β5−/−mice, expression levels did not vary. (C) Pros1 mRNA expression increases just before and at the time of peak phagocytosis in wt RPE/choroid and retina, respectively. In both tissue samples, a second peak occurs at night offset (retina) of just after (RPE/choroid). The 7.00 and 22.00 RPE/choroid peaks, as well as the phagocytosis retina peak, are lost in β5−/−mice, but median levels are not changed. (D) Corresponding protein quantification and representative immunoblots in the IPM of fellow eyes follows a combination of RPE/choroid and retina gene expression profiles depicted in (C). Results are in arbitrary units (a.u.) as means ± SDs, n = 3–8 independent samples; reference: wildtype sample at 8.00. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; two-way ANOVA with a Sidak post-test comparing wildtype and β5−/−samples at each time-point. Black bars: time-points during which lights were on (8.00–20.00); grey dotted bar, black tick: phagocytosis peak.

Article Snippet: Gas6 recombinant proteins (mouse, 986-GS), as well as anti-mouse antibodies raised in goats against Gas6 (AF986) and MFG-E8 (AF2805), were from R&D Systems (BioTechne, Noyal-Châtillon-sur-Seiche, France).

Techniques: Expressing, Western Blot, Gene Expression

Journal: International journal of molecular sciences

Article Title: Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

doi: 10.3390/ijms25126630

Figure Lengend Snippet: Figure 3. Gas6 is more expressed than Pros1, and ligands are more expressed in the retina than in the RPE/choroid. (A) Gas6 and Protein S (Pros1) mRNA expression profiles in RPE/choroid (green) and retina (orange) fractions of wildtype (wt) mice were compared at different times of day as indicated. Both ligands were more expressed in the retina than in the RPE/choroid. (B) Respective Gas6 (blue) and Pros1 (pink) mRNA expression profiles were compared in both the RPE/choroid and retina fractions of wt mice at different times of day as indicated. In both tissue types, Gas6 was much more expressed than Pros1. (A,B) Results are in arbitrary units (a.u.) as means ± SDs, n = 3–7 independent samples; references: RPE/choroid (A) or Pros1 (B) sample at 8.00. ** p < 0.01, *** p < 0.001, and **** p < 0.0001; two-way ANOVA with a Sidak post-test comparing wildtype and β5−/−samples at each time-point. Black bars: time-points during which lights were on (8.00–20.00); grey dotted bar, black tick: phagocytosis peak. (C) siRNA samples were used to downregulate the endogenous production of each ligand by RPE-J cells. Cells were then subjected to phagocytosis assays for 1.5 and 3 h as indicated. Decrease in Gas6 synthesis (blue bars) leads to diminished binding and internalization of POSs compared to control siRNA (Ctrl, white bars). Blocking the production of Protein S (Pros1, pink/purple bars) only slightly affects binding at 1.5 h. Adding both siRNAs has the same effect than adding the Gas6 siRNA alone. Targeting of both ligands’ production (purple bars) has the same effect as the decrease in Gas6 alone. Results of FITC/DAPI ratios are in arbitrary units (a.u.) expressed as means ± SDs, n = 5–6 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA with a Tukey post-test compared to each series corresponding control; reference: total phagocytosis (binding + internalization) for the control condition.

Article Snippet: Gas6 recombinant proteins (mouse, 986-GS), as well as anti-mouse antibodies raised in goats against Gas6 (AF986) and MFG-E8 (AF2805), were from R&D Systems (BioTechne, Noyal-Châtillon-sur-Seiche, France).

Techniques: Expressing, Binding Assay, Control, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

doi: 10.3390/ijms25126630

Figure Lengend Snippet: Figure 5. Gas6 and Protein S bind to different amino acids of MerTK Ig-like domains. Mutants target- ing ligand binding sites in MerTK Ig-like domains 1 (pink bars) and 2 (blue bars) were transfected in RPE-J and tested for their influence on POS binding (top left) and internalization (bottom left) when compared to non-mutated MerTK (black bar) with or without the addition of Gas6 and Protein S— alone or in combination—as indicated. The p.Gly122Arg (G122R, light pink bars) mutant significantly increases POS binding in DMEM while addition of Gas6 diminishes binding and addition of Protein S importantly increases internalization compared to the wt construct. Among the neighbor sites p.Thr140Ala (T140A) and p.Phe142Val (F142V), only p.Phe142Val (F142V) shows a slight increase in POS binding in the presence of Gas6. The p.Lys263Ile (K263I) mutant has a negative impact on both binding and internalization of POSs alone, as well as a positive effect on the internalization of POSs with Gas6. The p.Lys269Leu (K269L) mutant has almost no effect besides slightly less binding of POSs alone. When challenged with fluorescent beads (right bar graphs), no difference was observed in this study between the different clones. Results of FITC/DAPI ratios in arbitrary units (a.u.) are expressed as means ± SDs, with n = 4–6 independent experiments (POSs, left) or n = 3–4 independent experiments (beads, right). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; one-way ANOVA with a Tukey post-test compared to each series corresponding wildtype; reference: total phagocytosis (binding + internalization) for the control condition (WT). Significance brackets compare different ligand conditions for a single mutant.

Article Snippet: Gas6 recombinant proteins (mouse, 986-GS), as well as anti-mouse antibodies raised in goats against Gas6 (AF986) and MFG-E8 (AF2805), were from R&D Systems (BioTechne, Noyal-Châtillon-sur-Seiche, France).

Techniques: Ligand Binding Assay, Transfection, Binding Assay, Mutagenesis, Construct, Clone Assay, Control

Journal: Mediators of inflammation

Article Title: Growth Arrest-Specific 6 Enhances the Suppressive Function of CD4 + CD25 + Regulatory T Cells Mainly through Axl Receptor.

doi: 10.1155/2017/6848430

Figure Lengend Snippet: Figure 6: Gas6 modulates the Foxp3 and CTLA4 expression mainly through Axl receptor. (a–d) CD4+CD25+Tregs were treated with anti- Axl or anti-Mertk Abs or PBS in the presence of 100 ng/ml rmGas6. After 24 h of incubation, the expression of CTLA-4 and Foxp3 was determined by flow cytometry (𝑛= 4/group). ∗𝑃< 0.05 compared with the value for the Gas6 group, and #𝑃< 0.05 compared with the value for rmGas6+anti-Mertk group. (e–h) The expression of CTLA-4 and Foxp3 in Tregs with or without Axl knockout was determined by flow cytometry. ∗𝑃< 0.05 compared with the value for the Gas6 group.

Article Snippet: To investigate the effect of Gas6 on CD4+CD25+Tregs in vivo, healthy mice were administered 1, 3, or 6 μg/mouse of rmGas6 (8310-GS, R&D Systems, Minneapolis, MN) via tail vein.

Techniques: Expressing, Incubation, Flow Cytometry, Knock-Out

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Growth arrest-specific protein 6 (Gas6) pretreatment inhibits transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in lung epithelial cells (ECs). ( A – C ) LA-4 and ATII ECs were pretreated with 400 ng/mL Gas6 for 20 h prior to 10 ng/mL TGF-β1 treatment for 48 or 72 h. ( A ) Morphological changes in LA-4 ECs were examined by phase-contrast microscopy. Scale bars = 50 μm. Results are representative of three independent experiments. ( B ) Immunoblots of total cell lysates were performed with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies. Densitometry of the relative abundances of the indicated EMT markers. Alpha-tubulin was used as a control. ( C ) The amount of EMT markers’ mRNAs in cell lysates was analysed by real-time PCR and normalized to that of hypoxanthine phosphoribosyltransferase. Values represent the mean ± S.E. of three independent experiments. * P < 0.05; compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Microscopy, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Growth arrest-specific protein 6 (Gas6) pretreatment reduces epithelial-mesenchymal transition (EMT)-regulating transcription factor expression and blocks Smad-independent transforming growth factor (TGF)-β1 signalling in epithelial cells. ( A – C ) LA-4 and ATII epithelial cells (ECs) were pretreated with 400 ng/mL Gas6 20 h prior to 10 ng/mL TGF-β1 stimulation for 48 or 72 h. ( A , B ) The amounts of Snai1/2, Zeb1/2, and Twist1 mRNA were analysed by real-time PCR and normalized to that of hypoxanthine phosphoribosyltransferase ( Hprt ). ( C , D ) Representative immunoblots of LA-4 EC lysates were performed with anti-Snail1, -Zeb1, -total/phosphorylated ERK1/2, and -Akt protein antibodies. Beta-actin or alpha-tubulin was used as a loading control. Densitometric analysis of the indicated protein abundances. Data in all bar graphs are the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Cyclooxygenase (COX)-2 signaling is required for growth arrest-specific protein 6 (Gas6)-induced production of prostaglandin (PG)E 2 , PGD 2 , and their receptors. ( A – C ) LA-4 and primary alveolar type II (AT II) epithelial cells (ECs) were treated with 400 ng/mL Gas6 for the times indicated. ( A ) qPCR analysis of Cox2 and Cox1 mRNAs in cell lysates. ( B ) Representative immunoblots of LA-4 EC lysates were performed with anti-COX-2, -COX-1, or -α-tubulin antibodies. ( C ) PGE 2 or PGD 2 levels in conditioned media from LA-4 and AT II ECs were measured by enzyme immunoassay. ( D ) Immunoblots of total cell lysates were performed with anti-COX-2 antibodies in LA-4 ECs transfected with COX-2 specific or control siRNA for 6 h. Densitometric analysis of the COX-2 relative abundances. PGE 2 and PGD 2 levels in conditioned media from LA-4 ECs transfected with COX-2 specific or control siRNA for 6 h prior to treatment with 400 ng/mL Gas6 for 20 h were measured by EIA. ( E ) qPCR analysis of Ptger2, Ptger4, Dp1, and Dp2 mRNA in LA-4 ECs treated with 400 ng/mL Gas6 for the time indicated. ( F ) Immunoblot analysis of EP2, EP4, DP1, or DP2 in LA-4 cells. Densitometric analysis of the indicated receptor’ relative abundances. ( G ) qPCR analysis of Ptger2, Ptger4, Dp1, and Dp2 mRNA in LA-4 ECs transfected with COX-2 specific or control siRNA for 6 h prior to treatment with 400 ng/mL Gas6 for 20 h. ( H ) qPCR analysis of Ptger2, Ptger4, Dp1, and Dp2 mRNA in ATII ECs treated with 400 ng/mL Gas6 for the time indicated. Data in all bar graphs are the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Cyclooxygenase (COX)-2-derived signaling mediates growth arrest-specific protein 6 (Gas6)-induced epithelial-mesenchymal transition (EMT) inhibition. ( A – D ) LA-4 ECs were pretreated with 10 μM NS-398 1 h before 400 ng/mL Gas6 treatment for 20 h and then stimulated with 10 ng/mL TGF-β1 treatment for 72 h. ( A ) Morphological changes in the cells were examined by phase-contrast microscopy. Scale bars = 50 μm. ( B ) Immunoblots of total cell lysates were performed with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies. Densitometric analysis of the indicated EMT markers’ relative abundances. ( C , D ) Primary AT II cells were pretreated with 10 μM NS-398 1 h before 400 ng/mL Gas6 treatment for 20 h and then stimulated with 10 ng/mL TGF-β1 treatment for 72 h. qPCR analysis of the mRNAs of EMT markers and EMT-regulating transcription factors. ( E , F ) LA-4 ECs were transfected with COX-2-specific or control siRNAs for 6 h prior to treatment with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 72 h or the times indicated. Representative immunoblots of LA-4 EC lysates were performed with anti-E-cadherin, -N-cadherin, -α-SMA, -total/phosphorylated ERK1/2, and -Akt protein antibodies. Densitometric analysis of the indicated protein abundances. Data in all bar graphs are the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Inhibition, Microscopy, Western Blot, Transfection, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Prostaglandin (PG)E 2 and PGD 2 secretion inhibits growth arrest-specific protein 6 (Gas6)-induced epithelial-mesenchymal transition (EMT) via their receptors. ( A – D ) LA-4 and AT II epithelial cells (ECs) were stimulated with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 48 or 72 h with or without antagonists of EP2 (AH-6809), EP4 (AH-23848), DP1 (BW-A868C), or DP2 (BAY-u3405), each at a dose of 10 μM. ( A ) Representative immunoblots of total cell lysates were performed with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies. ( B ) qPCR analysis of the mRNAs of EMT transcription factors in LA-4 ECs. ( C , D ) qPCR analysis of the mRNAs of EMT markers and EMT transcription factors in primary AT II ECs. Values represent the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Activation of Axl or Mer mediates growth arrest-specific protein 6 (Gas6)-induced inhibition of COX-2 signaling and epithelial-mesenchymal transition (EMT) in LA-4 epithelial cells (ECs). ( A , B ) Immunoblot of total cell lysates were performed with anti-total/phosphorylated Axl and -Mer antibodies in LA-4 ECs treated with 400 ng/mL Gas6 for the times indicated. Densitometric analysis of the indicated protein abundances. ( C , D ) Immunoblots of total cell lysates were performed with anti-Axl, or -Mer antibodies in LA-4 ECs transfected with Axl, Mer, or control siRNA. Densitometric analysis of the indicated protein abundances. ( E – G ) LA-4 ECs were transfected with Axl, Mer, or control siRNAs for 48 h and then stimulated with 400 ng/mL Gas6. ( E , G ) qPCR analysis of the mRNAs of Cox2, Ptger2, Ptger4, Dp1, and Dp2 in LA-4 EC lysates 1 or 20 h after Gas6 stimulation. ( F ) PGE 2 and PGD 2 levels in conditioned media 20 h after Gas6 stimulation were measured by enzyme immunoassay. ( H – J ) LA-4 ECs were transfected with Axl, Mer, or control siRNAs for 48 h prior to treatment with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 72 h or the times indicated. ( H ) Representative immunoblots of total cell lysates with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies in the indicated samples. ( I ) qPCR analysis of the mRNAs of EMT transcription factors. ( J ) Representative immunoblots of total cell lysates with anti-total/phosphorylated ERK1/2 and -Akt protein antibodies. Values represent the mean ± S.E. of three independent experiments. * P < 0.05; compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Inhibition, Western Blot, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Growth arrest-specific protein 6 (Gas6)/Axl signaling inhibits migration and invasion of LA-4 and alveolar type II (AT II) epithelial cells (ECs) via prostaglandin (PG)E 2 and PGD 2 . ( A – D ) LA-4 and primary AT II ECs were stimulated with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 with or without antagonists of EP2 (AH-6809), EP4 (AH-23848), DP1 (BW-A868C), or DP2 (BAY-u3405), each at a dose of 10 μM, for 48 or 72 h. The quantification of migrated or invaded cells in Boyden chambers. ( E , F ) LA-4 ECs were transfected with Axl, Mer, or control siRNAs for 48 h prior to treatment with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 72 h. The cells were visualized by phase-contrast microscopy for the analysis of migratory in ( E ) left and invasive in ( F ) left abilities using Fn-coated Transwell and Matrigel-coated Transwell plates, respectively. Scale bars: 100 μm. Quantification of cells that migrated in ( E ) right or invaded in ( F ) right. Values represent the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Transfection, Control, Microscopy

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: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 2. Regulation of Axl shedding. L929 (A) and L929R (B) cells were treated with 200 ng/ml of PMA for different times, and the concentration of sAxl in cell-conditioned medium was evaluated by a specific ELISA. Untreated cells were used as controls. *, P 0.05 versus control samples. L929 (C) and L929R (D) cells were left untreated or treated with PMA for 2 h. Expression of membrane-bound Axl was evaluated by flow cytometry. (E and F) Cells were treated with PMA (200 ng/ml), Gas6 (50 ng/ml), IL-15 (50 ng/ml), TNF- (10 ng/ml), and LPS (10 ng/ml) for 2 h, and the concentration of sAxl in the culture medium was quantified by ELISA. Untreated (medium) cells were used as controls. **, P 0.01 versus control samples.

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Expressing, Membrane, Cytometry

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 7. sAxl is present in mouse serum and associates with Gas6. (A) Sera from C57BL/6, CH3, BALB/c, Axl/, Axl/, and Axl/

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 8. Immobilized Axl-Fc chimeric protein promotes cell migra- tion and induces the phosphorylation of Axl and PI3K. (A) L929R cells were plated onto Axl-Fc- or IL-3R–Fc (control)-coated six-well plates and allowed to grow in the presence of 10% FCS. After 18 h, a wound was created by scratching with a pipette tip (0 h). The cells were washed with PBS and incubated further to allow migration into the wounded area. Phase-contrast images of matched pairs of marked wound regions were taken 6 and 18 h later to assess cell migration. (B) L929R cells were serum starved for 4 h and incubated in Axl-Fc- coated wells for 15 min. Incubation in IL-3R–Fc (control)-coated wells or stimulation with 100 ng/ml of Gas6 was used as a negative or positive control, respectively. Cells were lysed, and Axl or PI3K was immunoprecipitated from the lysates with specific Abs. Precipitates were subjected to 10% SDS-PAGE and analyzed with anti-pTyr Abs. Detection of Axl or PI3K on the same blots was used as a loading control. The picture is representative of three independent experi- ments, all of which yielded highly comparable results. IP, immunopre- cipitation; WB, Western blotting.

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques: Phospho-proteomics, Control, Transferring, Incubation, Migration, Positive Control, Immunoprecipitation, SDS Page, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 9. Immobilized Axl-Fc chimeric protein induces cell migration and phosphorylation of Axl and PI3K in WT but not Axl/ MEFs. (A) MEFs were plated onto Axl-Fc- or IL-3R–Fc (control)-coated six-well plates. A confluent cell monolayer was wounded by scratching with a pipette tip (0 h). The cells were washed with PBS and incubated further to allow migration into the wounded area. Phase-contrast images of matched pairs of marked wound regions were taken 6 and 18 h later to assess cell migration. (B) MEFs were serum starved for 4 h and incubated in Axl-Fc-coated wells for 15 min. Incubation in IL-3R–Fc-coated (control) wells or stimulation with 100 ng/ml of Gas6 was used as a negative or positive control, respectively. Cells were lysed, and Axl or PI3K was immunoprecipitated from the lysates with specific Abs. Precipitates were subjected to 10% SDS-PAGE and analyzed with anti-pTyr Abs. Detection of Axl or PI3K on the same blots was used as a loading control. IP, immunoprecipitation; WB, Western blotting.

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques: Migration, Phospho-proteomics, Control, Transferring, Incubation, Positive Control, Immunoprecipitation, SDS Page, Western Blot