mouse anti gas6 igg2a  (R&D Systems)

Journal: bioRxiv

Article Title: Immune Niche Formation in Engineered Mouse Models Reveals Mechanisms of Tumor Dormancy

doi: 10.1101/2025.04.16.649000

Figure Lengend Snippet: A. Representative lung surface imaging of metastatic lesions from Tgfbr2 MyeKO and Tgfbr2 fl/fl (flox cont.) mice that received D2A1-H2B-GFP TVI for 12-14 days (left), and % dormant lesions (<8 cells) (middle) and total tumor lesions (right) at 12, 30, and 50 days after mice received TVI of tumor cells. n=7-9 mice per group. B. % 1-8 cell tumor lesions (left) and total tumor lesions (right) from the TSAE1+mHer2 TVI model, imaging on day 12. n=4 mice per group. C. % 1-8 cell tumor lesions (left) and total tumor lesions (right) from 4T1 orthotopic model, imaging on day 20. n=6 mice per group. D. Schematic design for sorting dormant (GFP+CVC Pos ) and proliferative D2A1-H2B-GFP (GFP+CVC Neg ) cells. E. Representative lung surface images of dormant (GFP+CVC Pos ) and proliferative lesions (GFP+CVC Neg ) from mice that received TVI of D2A1-H2B-GFP for 12 days. F. Representative Clearing-enhanced 3D (Ce3D) confocal imaging of lung tissues at 12 days after TVI of D2A1-H2B-GFP-mRuby-p27K cells. Green: H2B-GFP, Violet: mRuby-p27, and yellow: aSMA, with enlarged images in the middle panels; % 1-8 cell dormant lesions (lower left) and p27K+ dormant lesions (lower right). n=3 mice per group. G. Representative Imaging flow cytometry of P-p38 and GAS6 in dormant GFP+CVC Pos and proliferative GFP+CVC Neg D2A1 cells from Tgfbr2 MyeKO and flox cont. mice (left) and average of fluorescence intensity of P-p38 and GAS6 in GFP+CVC Pos and GFP+CVC Neg D2A1 and 4T1 cells (right). n=3-4 mice per group. H . Schematic design for the effect of Dox-induced myeloid-TβRII knockdown (KD) and myeloid-TβRII re-expression on dormant lesions. I-J. % dormant lesions from TβRII KD and re-expression after D2A1 TVI (I) and 4T1 orthotopic injection at day 12 (J). n=8 mice per group. All data are presented as mean ± s.e.m. P -values were derived from a two-tailed Student’s t-test and significance was determined by a P -value < 0.05.

Article Snippet: The cellular samples were then fixed, permeabilized, and stained with conjugated with P-p38-PE (Invitrogen, #12-9078-42), GAS6 (R&D systems, #AF986), and SLURP1(abbexa, #abx129448) antibodies for 40 minutes.

Techniques: Imaging, Flow Cytometry, Fluorescence, Knockdown, Expressing, Injection, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Immune Niche Formation in Engineered Mouse Models Reveals Mechanisms of Tumor Dormancy

doi: 10.1101/2025.04.16.649000

Figure Lengend Snippet: A. SLURP1 fluorescence intensity in dormant GFP+CVC Pos and proliferative GFP+CVC Neg tumor cells from Tgfbr2 MyeKO and flox cont. mice by Imaging flow cytometry, D2A1 (left) and 4T1 (right); n=4 biological independent experiments. B. % of SLURP1 + cells with P-p38 + and GAS6 + expression from dormant GFP+CVC Pos and proliferative GFP+CVC Neg tumor cells from Tgfbr2 MyeKO and flox cont. mice; n=4 four biological independent experiments. C. Representative lung surface imaging (left), and % dormant lesions from D2A1 tumor cells with Slurp1 KO and KD (right). n=5-12 mice per group. D. Western blot for SLURP1 overexpression (SLURP1-OE) in D2A1 tumor cells (D2A1-SLURP1-OE) (left), and % dormant lesions from mouse lungs at 12 days after TVI of D2A1-SLURP1-OE tumor cells (right). E. % dormant lesions from mouse lungs at day 30 after TVI of 4T1-SLURP1-OE tumor cells. n=9-10 mice per group. F. Representative images of D2A1 spheroid culture treated with recombinant SLURP1 (rSLURP1), and rSLURP1 plus an integrin activating antibody (IntA ab), as well as with rSLURP1 withdraw (left) and quantification of spheroid size (right). G-H. Flow cytometry analysis of P-FAK (G), P-p38, P-ERK, and the ratio of P-p38 to P-ERK (H) in 3D-cultured D2A1 cells treated with rSLURP1 or GST control. H. Schematic for SLURP1 mediated inhibition of tumor cell proliferation (created with BioRender.com ). All data are presented as mean ± s.e.m. P -values were derived from a two-tailed student t-test. Significance was determined by a P -value< 0.05.

Article Snippet: The cellular samples were then fixed, permeabilized, and stained with conjugated with P-p38-PE (Invitrogen, #12-9078-42), GAS6 (R&D systems, #AF986), and SLURP1(abbexa, #abx129448) antibodies for 40 minutes.

Techniques: Fluorescence, Imaging, Flow Cytometry, Expressing, Western Blot, Over Expression, Recombinant, Cell Culture, Control, Inhibition, Derivative Assay, Two Tailed Test

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