goat gas6  (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