goats against gas6 af986  (R&D Systems)

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:

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

doi: 10.1172/JCI30375

Figure Lengend Snippet: (A and B) Reduced number of erythroid progenitors in fetal liver of Gas6–/– mice, analyzed as the number of BFU-Es (A) or CFU-Es (B). n = 6 per group. *P < 0.05. (C and D) Reduced percentage of Ter-119+ nucleated cells (erythroblasts) in adult BM (C; *P = 0.02) and spleen (D; *P < 0.02) in Gas6–/– mice. The erythroblasts were quantified as the percent of all nucleated cells by flow cytometry. n = 9 per group. (E and F) Reduced number of erythroid progenitors in the BM of adult Gas6–/– mice, analyzed as the number of BFU-Es (E) or CFU-Es (F). n = 8 per group. *P < 0.05. Values are mean ± SEM.

Article Snippet: Goat polyclonal antibody against Gas6 (AF986; R&D Systems) was coated overnight at 4°C at 0.5 μg/ml.

Techniques: Flow Cytometry

Journal:

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

doi: 10.1172/JCI30375

Figure Lengend Snippet: WT and Gas6–/– mouse rbc were treated with PHZ to expose phosphatidylserine on their surface and then incubated with primary adherent BM-derived macrophages. (A and B) Macrophages in phase-contrast illumination are indicated by arrowheads; engulfed rbc are denoted by arrows. Phagocytosis was impaired in Gas6–/– mice (B) compared with WT mice (A). Scale bars: 20 μm. (C) The number of macrophages with 3 or more internalized rbc was reduced in Gas6–/– mice compared with WT mice (n = 250; mean ± SEM; *P = 0.001). Data were reproduced in 100% C57BL/6 background (not shown).

Article Snippet: Goat polyclonal antibody against Gas6 (AF986; R&D Systems) was coated overnight at 4°C at 0.5 μg/ml.

Techniques: Incubation, Derivative Assay

Journal:

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

doi: 10.1172/JCI30375

Figure Lengend Snippet: P values above the graphs denote overall genotypic difference during the entire period of analysis, as analyzed by ANOVA for repeated measurement statistics. *P < 0.05, standard t test. (A–D) Impaired erythropoiesis in Gas6–/– compared with WT mice in response to PHZ-induced hemolytic anemia. (A and B) Hematocrit levels (A) and reticulocyte indices (B) after 2 PHZ injections on days 0 and 1 (n = 8). Data were reproduced in 100% C57BL/6 background (Supplemental Figure 1). (C and D) Impaired erythropoiesis in Gas6–/– mice in response to autoimmune hemolytic anemia, induced by intraperitoneal injection of the 34-3C anti-mouse rbc monoclonal antibody (200 μg on day 0). (C) Hematocrit levels. (D) Reticulocyte indices (n = 8). (E) Impaired erythropoiesis in Gas6–/– splenectomized mice in response to injections of PHZ injections on days 0 and 1 (n = 6). (F) Hematocrit levels in Gas6–/– and WT mice after PHZ injections on days 0, 1 and 2 (n = 6). A hematocrit value of 11% (the lowest detectable hematocrit level in preterminal mice) was assigned to the mice that died in the course of the experiment. Values are mean ± SEM.

Article Snippet: Goat polyclonal antibody against Gas6 (AF986; R&D Systems) was coated overnight at 4°C at 0.5 μg/ml.

Techniques: Injection

Journal:

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

doi: 10.1172/JCI30375

Figure Lengend Snippet: Western blot analysis showing increased levels of Gas6 in cellular extracts and medium conditioned by UT7 human erythroid cells in response to 4 h stimulation with various concentrations of Epo. Total actin levels confirm the equal loading of cell protein extracts in each lane. Lanes showing Gas6 in cellular extracts were run on the same gel but were noncontiguous, as indicated by black lines.

Article Snippet: Goat polyclonal antibody against Gas6 (AF986; R&D Systems) was coated overnight at 4°C at 0.5 μg/ml.

Techniques: Western Blot

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: Goat polyclonal antibody against Gas6 (AF986; R&D Systems) was coated overnight at 4°C at 0.5 μg/ml.

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: Goat polyclonal antibody against Gas6 (AF986; R&D Systems) was coated overnight at 4°C at 0.5 μg/ml.

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) Western blot analysis of erythroblasts isolated from spleen 6 days after PHZ treatment from WT and Gas6–/– mice. (A) Total and tyrosine-phosphorylated (pTyr) EpoR expression in WT and Gas6–/– erythroblasts. (B) Akt phosphorylation in response to Gas6 alone (400 ng/ml), Epo alone (10 IU/ml), or a combination of both. Densitometric quantification of the amount of tyrosine-phosphorylated EpoR and total EpoR revealed a comparable ratio of pTyr-EpoR to EpoR in both genotypes (not shown).

Article Snippet: Goat polyclonal antibody against Gas6 (AF986; R&D Systems) was coated overnight at 4°C at 0.5 μg/ml.

Techniques: Western Blot, Isolation, Expressing, Phospho-proteomics

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: Goat polyclonal antibody against Gas6 (AF986; R&D Systems) was coated overnight at 4°C at 0.5 μg/ml.

Techniques: Saline, Control, Recombinant, Transgenic Assay

Journal:

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

doi: 10.1172/JCI30375

Figure Lengend Snippet: (A) Gas6 released by erythroblasts in response to Epo interacts with Gas6R on the cell surface, leading to signaling for cell survival and possible cell proliferation, maturation, and differentiation, and enhances EpoR signaling by activating PI3K and its effector Akt in these cells (autocrine effect). In addition, Gas6 favors adhesion of erythroblasts to fibronectin via VLA4/α4β1 integrin activation. (B) Gas6 acts as a bridging molecule between senescent rbc and Gas6Rs, driving engulfment of the bound senescent rbc. (C) Interaction of Gas6 and Gas6Rs downregulates the release of erythroid-inhibitory factors from macrophages in the erythroblastic island.

Article Snippet: Goat polyclonal antibody against Gas6 (AF986; R&D Systems) was coated overnight at 4°C at 0.5 μg/ml.

Techniques: Activation Assay