recombinant mouse gas6 protein  (R&D Systems)

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: bioRxiv

Article Title: EndoNB: A general strategy to study the internalization of cell surface proteins

doi: 10.1101/2025.06.08.658482

Figure Lengend Snippet: a , Structural alignment of the hybrid and β-I domains of wild-type and Alfa-tagged Integrin β1. b , Schematics showing the effect of 12G10 and Mab13 antibodies on integrin β1 activation state. These antibodies act by stabilizing the integrins in their active or inactive state. c , Quantification of integrin β1 uptake by flow cytometry using EndoNB in the presence of no antibodies (no treatment), or in the presence of 12G10 (activating), Mab13 (inactivating) or unrelated (anti integrin αvβ3 antibody). (n = 12, each n represents the median fluorescence of 2000-8000 cells). d , Structural alignment of the extracellular region of wild-type and Alfa-tagged transferrin receptor (TFR). Wild type: PDB 1suv, while Alfa-tagged was generated by AlphaFold 3. See for comparison of Alfa-tagged dimeric and monomeric TFR. e , Schematics showing the experimental design to test if transferrin receptor uptake is stimulated by the presence of transferrin. f , Quantification of transferrin receptor uptake for 15 or 30 minutes by flow cytometry using EndoNB in the presence of increasing concentrations of transferrin. (n = 4, each n represents the median fluorescence of 2000-8000 cells). See for quantification of transferrin-AlexaFluor488 signals at each condition. g , Structural alignment of the transmembrane and extracellular region of wild-type and Alfa-tagged AXL. Both structures generated with AlphaFold 3. h , Quantification of AXL uptake by flow cytometry using EndoNB in the absence or presence of the AXL ligand GAS6 (1ug/ml). (n = 4, each n represents the median fluorescence of 2000-8000 cells). See for comparison with the conditions with and without 3C. ns = non-significant, * p> 0.05, **** p> 0.0001. ANOVA with Tukey’s post hoc analysis.

Article Snippet: SNAP-JF646 substrate was a kind gift by Luke Lavis via the open chemistry team (jJanelia); Transferrin-Alexa Fluor 488 (Thermo Fisher scientific, #T13342); Phalloidin-iFluor 488 (AAT Bioquest, #23115); GAS6 recombinant protein (MedChemExpress via Nordic Biosite HY-P700724-20).

Techniques: Activation Assay, Flow Cytometry, Fluorescence, Generated, Comparison

Journal: bioRxiv

Article Title: EndoNB: A general strategy to study the internalization of cell surface proteins

doi: 10.1101/2025.06.08.658482

Figure Lengend Snippet: a , Structural alignment of the monomeric and dimeric extracellular region of wild-type and Alfa-tagged transferrin receptor (TFR, PDB). Wild type: PDB 1suv, while Alfa-tagged was generated by AlphaFold 3. b , Quantification of transferrin (AlexaFluor488) in each condition measured in . c , Quantification of AXL uptake by flow cytometry using EndoNB in the absence or presence of the AXL ligand GAS6 (1µg/ml) and also in the presence or not of 3C. (n = 4, each n represents the median fluorescence of 2000-8000 cells). ns = non-significant, ** p> 0.01, *** p> 0.001, **** p> 0.0001. ANOVA with Tukey’s post hoc analysis.

Article Snippet: SNAP-JF646 substrate was a kind gift by Luke Lavis via the open chemistry team (jJanelia); Transferrin-Alexa Fluor 488 (Thermo Fisher scientific, #T13342); Phalloidin-iFluor 488 (AAT Bioquest, #23115); GAS6 recombinant protein (MedChemExpress via Nordic Biosite HY-P700724-20).

Techniques: Generated, Flow Cytometry, Fluorescence

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: Cells were washed twice with PBS, and then incubated with either serum-free DMEM or full DMEM medium, with the addition of Recombinant Human Gas6 protein (R&D Systems, 885-GSB-050) or the equivalent amount of BSA.

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

Journal: Viruses

Article Title: Nano-Biomechanical Investigation of Phosphatidylserine-Mediated Ebola Viral Attachment via Human Gas6 and Axl

doi: 10.3390/v16111700

Figure Lengend Snippet: Characterization results of Gas6 interacting with Axl or PS on Ebola VLP. ( A ) Single molecule force measurement data are analyzed using the Bell–Evans model and the fitting results describe the Gas6–Axl (n = 451) and Gas6–VLP (n = 260) interactions as a relation between the unbinding force and loading rate. The fitting results also provide dissociation rates ( k 0 ) and reaction lengths ( γ ) of these two interactions to evaluate their binding affinity and bound complex energy barrier, which are listed as well. ( B ) Microscale thermophoresis (MST) analysis of the interaction between Gas6 and Axl (n = 7). The fitting result is used to determine the binding affinity in the form of dissociation constant (Kd). ( C ) The binding frequency comparison is used to show the interaction specificity between experiment groups and control groups. The binding frequency measurements are all conducted under the same conditions. All given error bars show the standard deviation. Significance is determined by an unpaired t -test. *: <0.05; **: <0.01; ns: not significant. n is the total sample number used for analysis.

Article Snippet: Human Gas6 protein was purchased from R&D systems (Minneapolis, MN, USA, catalog #885-GSB) and was expressed from mouse myeloma cell NS0, which contains AA Ala 49-Trp 678 (Accession #NP_000811).

Techniques: Binding Assay, Microscale Thermophoresis, Comparison, Control, Standard Deviation

Journal: Viruses

Article Title: Nano-Biomechanical Investigation of Phosphatidylserine-Mediated Ebola Viral Attachment via Human Gas6 and Axl

doi: 10.3390/v16111700

Figure Lengend Snippet: ( A ) Scheme shows the niche and the instrument settings during the measurement of VLP–Gas6–Axl (n = 427). ( B ) Single molecule force measurement result comparison between Gas6–Axl (n = 451), Gas6–VLP (n = 260), and VLP–Gas6–Axl (n = 427). Dissociation rate ( k 0 ) and reaction length ( γ ) for these interactions are shown. ( C ) The binding frequency comparison is used to show the interaction specificity between experiment groups and control groups. The binding frequency measurements are all conducted under the same conditions. All given error bars show the standard deviation. Significance is determined by an unpaired t -test. *: <0.05; **: <0.01; ns: not significant. n is the total sample number used for analysis.

Article Snippet: Human Gas6 protein was purchased from R&D systems (Minneapolis, MN, USA, catalog #885-GSB) and was expressed from mouse myeloma cell NS0, which contains AA Ala 49-Trp 678 (Accession #NP_000811).

Techniques: Comparison, Binding Assay, Control, Standard Deviation

Journal: Viruses

Article Title: Nano-Biomechanical Investigation of Phosphatidylserine-Mediated Ebola Viral Attachment via Human Gas6 and Axl

doi: 10.3390/v16111700

Figure Lengend Snippet: Single molecule force measurement result comparison to reveal the impact of calcium ion on interactions among VLP, Gas6, and Axl. ( A ) VLP–Gas6 interaction with (blue dashed line, n = 308) and without (blue solid line, n = 260) calcium ions present and their binding frequency comparison to examine the interaction specificity ( B ) VLP–Gas6–Axl interaction with (black dashed line, n = 397) and without (black solid line, n = 427) calcium ions present and their binding frequency comparison to examine the interaction specificity. ( C ) Gas6–Axl interaction with (red dashed line, n = 341) and without (red solid line, n = 451) calcium ions present and their binding frequency comparison to examine the interaction specificity. The binding frequency comparison is used to show the interaction specificity between experiment groups and control groups. The binding frequency measurements are all conducted under the same conditions. All given error bars show the standard deviation. Significance is determined by an unpaired t -test. *: <0.05; **: <0.01; ns: not significant. n is the total sample number used for analysis.

Article Snippet: Human Gas6 protein was purchased from R&D systems (Minneapolis, MN, USA, catalog #885-GSB) and was expressed from mouse myeloma cell NS0, which contains AA Ala 49-Trp 678 (Accession #NP_000811).

Techniques: Comparison, Binding Assay, Control, Standard Deviation

Journal: Viruses

Article Title: Nano-Biomechanical Investigation of Phosphatidylserine-Mediated Ebola Viral Attachment via Human Gas6 and Axl

doi: 10.3390/v16111700

Figure Lengend Snippet: Comparison and summary of all single molecule force measurement results in this study to reveal the binding mechanism among VLP, Gas6, and Axl biomechanically and the impact of calcium ions on binding strengths and binding affinities.

Article Snippet: Human Gas6 protein was purchased from R&D systems (Minneapolis, MN, USA, catalog #885-GSB) and was expressed from mouse myeloma cell NS0, which contains AA Ala 49-Trp 678 (Accession #NP_000811).

Techniques: Comparison, Binding Assay

Journal: Heliyon

Article Title: Cabozantinib prevents the progression of metabolic dysfunction-associated steatohepatitis by inhibiting the activation of hepatic stellate cell and macrophage and attenuating angiogenic activity

doi: 10.1016/j.heliyon.2024.e38647

Figure Lengend Snippet: Effect of cabozantinib on GAS6/AXL signaling in LX-2 cells. (A) Serum Gas6 level in the experimental rats. (B) Hepatic mRNA expression of Axl in the experimental rats. (C) Western blotting for the effect of cabozantinib (CBZ) (0−50 nM) on the phosphorylation of AXL and AKT in rGAS6 (500 ng/mL)-stimulated LX-2 cells. Whole images of blotting were shown in in Effect of CBZ (0−50 nM) on rGAS6 (500 ng/mL)-induced LX-2 proliferation. (E) Inducible effect of rGAS6 (500 ng/mL) on the mRNA expression of profibrogenic markers (ACTA2, COL1A1, TIMP1 and CTGF) in LX-2 cells. (F) Effect of CBZ (0−50 nM) on the mRNA expression of profibrogenic markers in rGAS6 (500 ng/mL)-stimulated LX-2 cells. Gapdh/GAPDH was used as an internal control for qRT-PCR (B, E and F). Actin was used as an internal control for western blotting (C). Quantitative values are indicated as fold changes to the values of CS group (B), the value of each group at 0 h (D), the value at 0 h (E) and the value of rGAS6 (−)/CBZ (−) group (F). Data are the mean ± SD (n = 8; A and B, n = 6; D−F). a, aa : P < 0.05, 0.01 vs CS group (A and B), rGAS6 (−)/CBZ (−) group at 72 h (D), 0 h group (E), rGAS6 (−)/CBZ (−) group (F), b, bb : P < 0.05, 0.01 vs CD + Veh group (A and B), rGAS6 (+)/CBZ (−) group at 72 h (D), rGAS6 (+)/CBZ (−) group (F) significant difference between groups by Student's t-test. CS, CSANFD-fed and vehicle-treated group; CD + Veh, CDAHFD-fed and vehicle-treated group; CD + CBZ-L, CDAHFD-fed and cabozantinib (1 mg/kg)-treated group; CD + CBZ-H, CDAHFD-fed and cabozantinib (2 mg/kg)-treated group.

Article Snippet: LX-2 cells were treated with recombinant human GAS6 (rGAS6; 500 ng/mL; R&D Systems) or recombinant human VEGF 165 (VEGFA; 10 ng/mL; Peprotech, Waltham, MA, USA) to stimulate cell proliferation and profibrogenic activity.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR