recombinant mouse gas6 protein  (R&D Systems)

Journal: Journal of Nanobiotechnology

Article Title: Microglia-specific interleukin-4 delivery by engineered extracellular vesicles restores inner blood-retinal barrier in diabetic retinopathy via GAS6-MERTK pathway

doi: 10.1186/s12951-025-03976-w

Figure Lengend Snippet: IL4@CHHSSSARC-EV promoted microglial phagocytosis via GAS6-MERTK pathway. A Representative immunofluorescence staining images of IBA1 and C-C3 on retinal flat mounts from OIR mice treated with PBS or IL4@CHHSSSARC-EV. Scale bar: 100 μm. Enlarged: 50 μm. B qPCR analysis of GAS6 and TAM systems (Axl, MERTK, Tyro3) mRNA levels in M1 microglia exposed to PBS or IL4@CHHSSSARC-EV ( n = 3). C Concentrations of GAS6 in the supernatant of M1 microglia after exposed to PBS or IL4@CHHSSSARC-EV by ELISA ( n = 3). D Representative western blot images and quantitative analysis of protein expression of p-MERTK and MERTK after M1 microglia were exposed to PBS or IL4@CHHSSSARC-EV ( n = 3). E Representative immunofluorescence staining images of IBA1 and C-C3 on retinal flat mounts from OIR mice treated with IL4@CHHSSSARC-EV or IL4@CHHSSSARC-EV plus UNC2025. Scale bar: 100 μm. Enlarged: 50 μm. F Representative western blot images and quantitative analysis of protein expression of p-MERTK and MERTK after M1 microglia were exposed to IL4@CHHSSSARC-EV or IL4@CHHSSSARC-EV plus UNC2025 ( n = 3). G Representative immunofluorescence staining images of IB4 in retinas from OIR mice treated with IL4@CHHSSSARC-EV or IL4@CHHSSSARC-EV plus UNC2025 ( n = 5). Scale bar: 500 μm. White region indicated neovascular area and yellow-circulated region marked avascular area. H Quantitative analysis of neovascular area and avascular area of G . * P < 0.1, ** P < 0.01, *** P < 0.001

Article Snippet: The concentration of GAS6 was determined by the mouse GAS6 ELISA kit (EK2384, Multi Sciences).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Spatio-temporal dynamics of the fibrotic niche in cardiac repair

doi: 10.1101/2024.11.10.622609

Figure Lengend Snippet: A , Spatial maps showing MP and FB subtypes in the wound (LV edge in sham). B , day 7 neighborhoods surrounding Postn+Thbs4+ myoFB, with MP highlighted in blue. C , spatial gene factor co-variations of MP neighborhood in day 7 heart. Circle size scales linearly with −log 10 (p-value), and circle color indicates ridge regression coefficients. D , genes positively and negatively associated with gene factor 3 (Fa3) of MP and Fa2 of FB, respectively. Highlighted genes in MP-positive-Fa3: secreted ligands; MP-negative-Fa3: early proinflammatory/IFN pathway; FB-positive-Fa2: ECM genes, FB-negative-Fa2: cell cycle. E , pathways negatively associated with FB Fa2 (BioPlanet 2019 database). F , FB UMAP with inferred cell fate transition and cell cycle G2M gene score (upper), and time points post-lesion. G , boxplot comparing log2-transformed aggregated G2M gene expression of FB over time. H , CellChat ligand-receptor interaction prediction between MP (signal senders) and FB subtypes (signal receivers) across day 3–28. I , immune cell UMAP showing Gas6 and Pros1 expression, and FB UMAP showing Axl expression. J , multiplexed-IF staining of day 7 post-LAD IZ. Dotted lines highlight CD45+ GAS6+ PROS1+ immune cells contacting myoFB (CD140a+). K – M , mouse cardiac myoFB culturing experiment (Methods). Cultured myoFB were harvested for IF staining with αSMA and Mki67 ( K ), quantification of % Mki67+ nuclei (DAPI+) in αSMA+ cells (myoFB) ( l ), as well as qRT-PCR quantification of senescence marker gene Glb1 (β-gal) ( M ). N , CellChat prediction for FB (senders) and MP subtypes (receivers), across day 3-28. O , UMAPs of FB (left) and immune (right) clusters plotting SEMA3 family and their receptor’s gene expression. P and Q , mouse BMDM culturing. BMDM were exposed to BSA/SEMA3D for 72 hours, then harvested for H3P IF ( P ) with intensity quantification ( Q ). For ( L ), ( M ), ( P ), ( Q ), statistical significance was calculated by unpaired two-tailed t-test for two experimental groups. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns, not significant.

Article Snippet: The next day, the medium was removed and cells were washed twice with PBS, before being incubated with serum-free DMEM with Recombinant Mouse GAS6 protein (0.05– 1000ng/mL, R&D Systems, 986-GS-025/CF), Recombinant Mouse Protein S/PROS1 (100ng/mL, R&D Systems, 9740-PS-050/CF), or an equivalent amount of BSA.

Techniques: Transformation Assay, Expressing, Staining, Cell Culture, Quantitative RT-PCR, Marker, IF-P, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: Microglia-specific interleukin-4 delivery by engineered extracellular vesicles restores inner blood-retinal barrier in diabetic retinopathy via GAS6-MERTK pathway

doi: 10.1186/s12951-025-03976-w

Figure Lengend Snippet: IL4@CHHSSSARC-EV promoted microglial phagocytosis via GAS6-MERTK pathway. A Representative immunofluorescence staining images of IBA1 and C-C3 on retinal flat mounts from OIR mice treated with PBS or IL4@CHHSSSARC-EV. Scale bar: 100 μm. Enlarged: 50 μm. B qPCR analysis of GAS6 and TAM systems (Axl, MERTK, Tyro3) mRNA levels in M1 microglia exposed to PBS or IL4@CHHSSSARC-EV ( n = 3). C Concentrations of GAS6 in the supernatant of M1 microglia after exposed to PBS or IL4@CHHSSSARC-EV by ELISA ( n = 3). D Representative western blot images and quantitative analysis of protein expression of p-MERTK and MERTK after M1 microglia were exposed to PBS or IL4@CHHSSSARC-EV ( n = 3). E Representative immunofluorescence staining images of IBA1 and C-C3 on retinal flat mounts from OIR mice treated with IL4@CHHSSSARC-EV or IL4@CHHSSSARC-EV plus UNC2025. Scale bar: 100 μm. Enlarged: 50 μm. F Representative western blot images and quantitative analysis of protein expression of p-MERTK and MERTK after M1 microglia were exposed to IL4@CHHSSSARC-EV or IL4@CHHSSSARC-EV plus UNC2025 ( n = 3). G Representative immunofluorescence staining images of IB4 in retinas from OIR mice treated with IL4@CHHSSSARC-EV or IL4@CHHSSSARC-EV plus UNC2025 ( n = 5). Scale bar: 500 μm. White region indicated neovascular area and yellow-circulated region marked avascular area. H Quantitative analysis of neovascular area and avascular area of G . * P < 0.1, ** P < 0.01, *** P < 0.001

Article Snippet: The concentration of GAS6 was determined by the mouse GAS6 ELISA kit (EK2384, Multi Sciences).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Spatio-temporal dynamics of the fibrotic niche in cardiac repair

doi: 10.1101/2024.11.10.622609

Figure Lengend Snippet: A , Spatial maps showing MP and FB subtypes in the wound (LV edge in sham). B , day 7 neighborhoods surrounding Postn+Thbs4+ myoFB, with MP highlighted in blue. C , spatial gene factor co-variations of MP neighborhood in day 7 heart. Circle size scales linearly with −log 10 (p-value), and circle color indicates ridge regression coefficients. D , genes positively and negatively associated with gene factor 3 (Fa3) of MP and Fa2 of FB, respectively. Highlighted genes in MP-positive-Fa3: secreted ligands; MP-negative-Fa3: early proinflammatory/IFN pathway; FB-positive-Fa2: ECM genes, FB-negative-Fa2: cell cycle. E , pathways negatively associated with FB Fa2 (BioPlanet 2019 database). F , FB UMAP with inferred cell fate transition and cell cycle G2M gene score (upper), and time points post-lesion. G , boxplot comparing log2-transformed aggregated G2M gene expression of FB over time. H , CellChat ligand-receptor interaction prediction between MP (signal senders) and FB subtypes (signal receivers) across day 3–28. I , immune cell UMAP showing Gas6 and Pros1 expression, and FB UMAP showing Axl expression. J , multiplexed-IF staining of day 7 post-LAD IZ. Dotted lines highlight CD45+ GAS6+ PROS1+ immune cells contacting myoFB (CD140a+). K – M , mouse cardiac myoFB culturing experiment (Methods). Cultured myoFB were harvested for IF staining with αSMA and Mki67 ( K ), quantification of % Mki67+ nuclei (DAPI+) in αSMA+ cells (myoFB) ( l ), as well as qRT-PCR quantification of senescence marker gene Glb1 (β-gal) ( M ). N , CellChat prediction for FB (senders) and MP subtypes (receivers), across day 3-28. O , UMAPs of FB (left) and immune (right) clusters plotting SEMA3 family and their receptor’s gene expression. P and Q , mouse BMDM culturing. BMDM were exposed to BSA/SEMA3D for 72 hours, then harvested for H3P IF ( P ) with intensity quantification ( Q ). For ( L ), ( M ), ( P ), ( Q ), statistical significance was calculated by unpaired two-tailed t-test for two experimental groups. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns, not significant.

Article Snippet: The following antibodies were used: mouse PDGFRa (R&D Systems, AF1062-SP), rat CD45 (Biolegend, 103108), goat GAS6 (R&D Systems, AF986-SP), rabbit PROS1 (Invitrogen, PA5-106880), goat AXL (R&D Systems, AF854-SP), mouse BMP-7 (NovusBio, NBP2-52425), rabbit GATA3 (Cell Signaling Technology, 5852T), goat KIT (R&D Systems, AF1356-SP).

Techniques: Transformation Assay, Expressing, Staining, Cell Culture, Quantitative RT-PCR, Marker, IF-P, Two Tailed Test

Journal: Nature Communications

Article Title: Interaction between subventricular zone microglia and neural stem cells impacts the neurogenic response in a mouse model of cortical ischemic stroke

doi: 10.1038/s41467-024-53217-1

Figure Lengend Snippet: a DCX immunostaining (green) in combination with RFP (red) and GFAP (blue) labeling in the SVZ and the lesion penumbra 7 days after PT. White boxes indicate enlarged areas showing the SVZ showing DCX and RFP expression, and IMARIS reconstruction of the lesion penumbra, with few DCX + migratory neuroblasts (arrowhead) ( n = 1). The lesion core is indicated by a dotted line. Scales (l to r): 750, 100, 260 µm. b DCX immunostaining (green) with RFP (red) and CD68 (gray) labeling in the SVZ 1 day after PT, indicating early SVZ microglia activation after cortical stroke ( n = 1). Scale, 15 µm. c scRNA-Seq and analysis of SVZ NSPCs and microglia after PT. d UMAP representation of four NSPC clusters from Supplementary Fig. i. e NSPC cluster-specific gene expression defining their localization along the UMAP of Supplementary Fig. 1i. f UMAP visualization of fate-mapped NSPCs captures differentiation from quiescent type B cells to neuroblasts after PT. g Pseudotime progression along the NSPC differentiation trajectory after PT. Black arrows indicate type B cell activation and differentiation at day 1 after PT. h Ascl1 + proliferative type C cells co-express Gas6 after PT. i Immunolabeling for Gas6 (green) and Ki67 (red) in the SVZ 1 day after PT. Dashed boxes indicate magnifications of Gas6 − Ki67 + (top) and Gas6 + Ki67 + cells (bottom, asterisk). Scales, 24(left), 13 µm (magnified images). Right, quantification ( n = 7 mice). j Immunolabeling for DCX (green) and ApopTag (red) in the SVZ 7 days after PT. Dashed boxes indicate magnifications of ApopTag−DCX + (top) and ApopTag + DCX + cells (bottom, asterisk) of control and 7 days after PT, respectively. Scales, 50 (left), 16 µm (magnified images). Right, quantification ( n = 4 mice, uninjured; n = 9 mice, PT D7). k UMAP representation of five distinct microglial clusters obtained after subcluster analysis of the identified microglial cluster in Supplementary Fig. 1i. l Quantification of microglial cluster proportions. m UMAP plots of expression of core signature genes of SVZ microglial cluster. All graphs show the mean ± SEM. *** P < 0.001, **** P < 0.0001, unpaired Student’s t tests. RMS, rostral migratory stream; SVZ, subventricular zone. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were rabbit anti-Iba1 (1:500, Wako, 019-19741), goat anti-CD31 (1:500, R&D Systems, AF3628-SP), guinea pig anti-Doublecortin (1:1000, Millipore, AB2253), rat anti-GFAP (1:2000, Invitrogen, 13-0300), goat anti-GFP (1:2000, Abcam, ab5450), rabbit anti-GFP (1:2000, Abcam, ab290), sheep anti-Fibrinogen (1:500, US Biological, F4203-02F), goat anti-Nestin (1:200, Santa Cruz, sc-21249), goat anti-Nestin (1:500, Antibodies-Online, ABIN188165), rabbit anti-TMEM119 (1:500, Abcam, ab209064), rat anti-CD68 (1.200, Biorad, MCA1957), rabbit anti-C1qb (1:200, Abcam, ab182451), rabbit anti-P2ry12 (1:500, Sigma Life Science, HPA014518), rabbit anti-Sox2 (1.2000, Abcam, ab97959), rabbit anti-Mash-1/Achaete-scute (1:500, Abcam, ab74065), rabbit anti-RFP (1:2000, Rockland, 600-401-379), mouse anti-iNOS (1:200, BD Bioscience, 610329), rabbit anti-Gas6 (1:25, R&D Systems, AF986), rat anti-Ki67 (1:200, Invitrogen, 14-5698-82), goat anti-ApoE (1:50, Invitrogen, PA1-26902), rabbit anti-ApoE (1:50, Abcam, ab183597), rat anti-CD45 (1:100, BD Pharmingen, 553079), mouse anti-EGFR (1:100, Merck Millipore, 05-1047), rabbit anti-Lrp8 (1:50, ThermoFisher, bs-6651R), rabbit anti-NeuN (1:500, Abcam, ab177487), mouse anti-Olig2 (1:200, Merck Millipore, MABN50), rabbit anti-S100β (1:2000, Abcam, ab868).

Techniques: Immunostaining, Labeling, Expressing, Activation Assay, Immunolabeling, Control