Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α (SIRPA) expression is downregulated in dilated cardiomyopathy (DCM) human hearts and aortic banding (AB)-operated mouse hearts. A and B, Protein levels of β-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), and SIRPA in samples from donor hearts and DCM hearts (n=3; *P<0.05 vs donor hearts). C and D, Protein levels of β-MHC, ANP, and SIRPA in samples from wild-type mice at the indicated times after sham or AB surgery (n=3; *P<0.05 vs sham). E and F, Protein levels of β-MHC, ANP, and SIRPA in samples from neonatal rat cardiomyocytes treated with angiotensin II (Ang II; 1 μmol/L) or phenylephrine (PE; 100 μmol/L) for 48 h (n=3; *P<0.05 vs PBS). Representative blots (A, C, and E) and quantitative results (B, D, and F). n indicates number of independent experiments.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Expressing

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α (SIRPA) deficiency augments pressure overload–induced cardiac hypertrophy and fibrosis. A, Images of heart sections stained with H&E and WGA from wild-type (WT) and knockout mice 4 weeks after sham or aortic banding (AB) surgery (n=6–10 mice per experimental group). B, Statistical results for the cell sectional area (n=>100 cells). C–E, Statistical results for the ratios of (C) heart weight (HW)/body weight (BW), (D) lung weight (LW)/BW, and (E) HW/tibia length (TL) in the indicated groups (n=13–15 mice per experimental group). F, Images of heart sections stained with picrosirius red from WT and knockout mice 4 weeks after sham or AB surgery (n=6–10 mice per experimental group). G, Statistical results for the left ventricular (LV) collagen volume (%; n=>40 fields). *P<0.05 vs WT/sham; #P<0.05 vs WT/AB.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Staining, Knock-Out

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α overexpression mitigates aortic banding (AB)-induced cardiac remodeling. A, Images of heart sections stained with H&E and WGA from nontransgenic (NTG) and transgenic (TG) mice 8 weeks after sham or AB surgery (n=6 mice per experimental group). B, Statistical results for the cell sectional area (n=>100 cells). C–E, Statistical results for the ratios of (C) heart weight (HW)/body weight (BW), (D) lung weight (LW)/BW, and (E) HW/tibia length (TL) in the indicated groups (n=14–15 mice per experimental group). F, Images of heart sections stained with PSR from NTG and TG mice 8 weeks after sham or AB surgery (n=6 mice per experimental group). G, Statistical results for the left ventricular collagen volume (%; n=>40 fields). *P<0.05 vs NTG/sham; #P<0.05 vs NTG/AB.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Over Expression, Staining, Transgenic Assay

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Signal regulatory protein-α (SIRPA) inhibits the Toll-like receptor 4 (TLR4) signaling pathway. A and B, Protein expression levels of TLR4 are upregulated in the SIRPA knockout mice but downregulated in the SIRPA transgenic mice 2 weeks after aortic banding (AB) surgery (n=3; *P<0.05 vs wild-type [WT] or nontransgenic (NTG)/sham; #P<0.05 vs WT or NTG/AB). Representative blots (top); quantitative results (bottom). C, Representative colocalization images of SIRPA and TLR4 in HEK293T cells. Red, TLR4; green, SIRPA; blue, nucleus. D, SIRPA interacts with TLR4. Western blot with the Flag or Myc antibody after the coimmunoprecipitation of TLR4 from HEK293T whole-cell lysates using the Flag antibody (left). Western blot with the Flag or Myc antibody after the coimmunoprecipitation of SIRPA from HEK293T whole-cell lysates using the Myc antibody (right). E and F, Phosphorylation and total protein levels of IκBα and P65 in samples from (E) WT and SIRPA knockout mice and (F) NTG and SIRPA transgenic mice 2 weeks after AB surgery (n=3; *P<0.05 vs WT or NTG/AB). Representative blots (top); quantitative results (bottom). n indicates number of independent experiments.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Expressing, Knock-Out, Transgenic Assay, Western Blot, Phospho-proteomics

Journal: Hypertension

Article Title: Signal Regulatory Protein-α Protects Against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling

doi: 10.1161/HYPERTENSIONAHA.113.01506

Figure Lengend Snippet: Disruption of Toll-like receptor 4 (TLR4) rescued the adverse effect of signal regulatory protein-α (SIRPA) deficiency on cardiac hypertrophy. A, Images of heart sections stained with H&E and WGA from the indicated groups (n=5 mice per experimental group). B, Statistical results for the cell sectional area (n=>100 cells). C–E, Statistical results for the ratios of (C) heart weight (HW)/body weight (BW), (D) lung weight (LW)/BW, and (E) HW/tibia length (TL) in the indicated groups (n=11 mice per experimental group). F and G, Phosphorylation and total protein levels of IκBα and P65 in samples from the indicated groups 4 weeks after aortic banding(AB) surgery (n=3 independent experiments). Representative blots (F); quantitative results (G). DKO indicates double knockout.

Article Snippet: 16 Cardiac-specific SIRPA transgenic (TG) mice were generated by cloning full-length mouse SIRPA cDNA (OriGene; MC218099) downstream of the α-myosin heavy chain (α-MHC) promoter.

Techniques: Disruption, Staining, Phospho-proteomics, Double Knockout

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) Existing model by which SIRPα suppresses phagocytosis by interacting in trans with CD47 on target cells. See text for details. The 3 Ig-like domains of SIRPα (1 IgV and 2 IgCs) and the single Ig-V domain of CD47 are shown as ellipses. Mβs, macrophages. ( B ) Depiction of SIRPα variants and their functional characteristics. SIRPα FFFF contained substitution of tyrosine (Y)-to-phenylalanine (F) substitution at Y436, 460, 477, and 501; SIRPα ΔIC lacked most of the cytoplasmic domain of SIRPα, ending with arginine 401; SIRPα T96V carried a threonine (T)-to-valine (V) mutation at position 96 (shown by lavender star), which abolishes CD47-binding; SIRPα T96V,FFFF had the T96V and FFFF mutations; SIRPα T96V,ΔIC had the T96V and the ΔIC mutations. KO, knock-out. ITIM, immunoreceptor tyrosine-based inhibitory motif. ( C to G ) SIRPα variants or empty vector were expressed in SIRPα KO BMDMs and tested. Wild-type (WT) BMDMs were used as control. ( C ) Schematic representation of assays performed. Fc, fragment crystallizable. ( D ) Flow cytometry analyses of SIRPα expression and CD47-binding. APC, allophycocyanin. AF647, Alexa fluor 647. ( E and F ) Representative ( E ) and compiled data ( F ) of pHrodo-based phagocytosis assays using L1210 derivatives expressing Tac and opsonized with Tac monoclonal antibody (mAb) 7G7, as targets. Positive cells with percentages are boxed. G , Efficiency of phagocytosis inhibition in SIRPα KO BMDMs expressing or not the indicated SIRPα variants was calculated using the values in ( F ). SIRPα KO expressing WT SIRPα or empty vector displayed 100% and 0% inhibition efficiency, respectively. All data are means ± s.e.m., **** p < 0.0001. Results in ( D and E ) are representative of 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that are representative of 3 experiments. Results in ( F and G ) are pooled from a total of 6 mice studied in 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that involved 3 mice in 3 experiments. Each symbol in ( F ) represents one mouse.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Functional Assay, Mutagenesis, Binding Assay, Knock-Out, Plasmid Preparation, Control, Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) Immunoprecipitation followed by mass spectrometry of SIRPα-associated proteins. ( A ) Schematic representation of assay. ( B ) Plasma membrane-associated proteins found in SIRPα immunoprecipitates from WT BMDMs, but not from SIRPα KO BMDMs. c , Co-immunoprecipitation assay of SIRPα, CD18 and CD11b in WT and SIRPα KO BMDMs. IP, immunoprecipitation. Abs, antibodies. ( D to F ) FRET assays. ( D ) Schematic representation of FRET assay in HEK293T cells. ( E and F ) Representative confocal microscopy images ( E ) and compiled data ( F ) of FRET assays with donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of control (Ctrl) IgG, CD18 mAb GAME-46 or CD11b mAb 5C6. Yellow to purple spectrum denotes strong to weak FRET. DIC, differential interference contrast. Scale bars, 5 μm. ( G and H ) LUV-FRET assay. ( G ) Schematic representation of LUV-FRET assay. ( H ), Time-course of donor-labeled SIRPα fluorescence intensity after addition of acceptor-labeled CD18 or CD11b, monitored with a real-time plate reader. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( C , E and H ) are representative of 3 independent experiments. Results in ( B and F ) are pooled from a total of 3 independent experiments. Each symbol in ( F ) represents one cell.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Immunoprecipitation, Mass Spectrometry, Clinical Proteomics, Membrane, Co-Immunoprecipitation Assay, Confocal Microscopy, Labeling, Control, Fluorescence

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) FRET assays with SIRPα and SIRPβ1a. ( A ) A schematic representation of SIRPα and SIRPβ1a, with their 1 IgV domain and 2 IgC domains, is depicted. ( B ) Compiled data of 3 independent experiments using donor-labeled SIRPα or SIRPβ1a, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( C and D ) FRET assays using SIRPα IgV domain. ( C ) A schematic representation of a SIRPα variant having only the IgV domain is shown. ( D ) Compiled data of 3 independent experiments using donor-labeled SIRPα IgV, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( E - H ) FRET assays using SIRPα variants carrying non-conserved residues from SIRPβ1a. ( E and G ) Schematic representations of SIRPα variants. ( F and H ) Compiled data from 3 independent experiments using donor-labeled SIRPα variants, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( I to K ) Proximity ligation assay (PLA) of SIRPα and CD18 in BMDMs expressing or not the indicated SIRPα variants. (I) Flow cytometry analyses of SIRPα expression. ( J and K ) Representative confocal microscopy images ( J ) and compiled data from 3 independent experiments ( K ) of PLA for SIRPα and CD18. Scale bar, 10 μm. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( I and J ) are representative of 3 independent experiments. Results in ( B , D , F , H and K ) are pooled from 3 independent experiments. Each symbol in ( B , D , F , H and K ) represents one cell or mouse.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Labeling, Variant Assay, Proximity Ligation Assay, Expressing, Flow Cytometry, Confocal Microscopy

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis

Journal: Cancer Science

Article Title: Characterization of myeloid signature genes for predicting prognosis and immune landscape in Ewing sarcoma

doi: 10.1111/cas.15688

Figure Lengend Snippet: The primer sequences of the candidate genes

Article Snippet: All tissue slices were subjected to a series of steps such as dewaxing, antigen retrieval, blocking, primary antibody incubation, and secondary antibody incubation, including CTSD (Proteintech, 21327‐1‐AP), SIRPA (Affinity, AF0253), FN1 (Proteintech, 15613‐1‐AP), CD8 (Proteintech, 66868‐1‐lg), CD206 (Proteintech, 60143‐1‐lg), CD66b (Abcam, ab300122), and CD57 (Servicebio, GB113461).

Techniques: Sequencing

Journal: Cancer Science

Article Title: Characterization of myeloid signature genes for predicting prognosis and immune landscape in Ewing sarcoma

doi: 10.1111/cas.15688

Figure Lengend Snippet: In vitro validation of CTSD, SIRPA, and FN1 expression. A, Western blotting showing protein expression levels of CTSD, SIRPA, and FN1 in Ewing sarcoma (EWS) cell lines and normal controls; B‐D, Quantitative real‐time PCR showing the relative mRNA expression levels of CTSD, SIRPA, and FN1 in EWS cell lines and normal controls. E‐G, Representative immunohistochemistry staining of CTSD, SIRPA, and FN1 in EWS tissue and matched para‐cancer. H‐J, Quantitative analysis of the positive rate of pathological stained sections for these three genes. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: All tissue slices were subjected to a series of steps such as dewaxing, antigen retrieval, blocking, primary antibody incubation, and secondary antibody incubation, including CTSD (Proteintech, 21327‐1‐AP), SIRPA (Affinity, AF0253), FN1 (Proteintech, 15613‐1‐AP), CD8 (Proteintech, 66868‐1‐lg), CD206 (Proteintech, 60143‐1‐lg), CD66b (Abcam, ab300122), and CD57 (Servicebio, GB113461).

Techniques: In Vitro, Biomarker Discovery, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 1 | Engineering and characterization of receptor-based Axl antagonists. (a) Axl’s extracellular domain consists of two Ig-like domains containing high- and low-affinity Gas6 binding sites, followed by two fibronectin type III domains. Binding of Gas6 to Axl leads to receptor dimerization and activation of downstream signaling. Axl decoy receptors sequester Gas6, preventing activation of the Axl signaling cascade. (b) Overlaid flow cytometry dot plots representing binding of yeast-displayed wild-type Axl Ig1 (red) and unsorted Axl Ig1 library (blue) to 10 nM Gas6 (y axis) and expression levels on the yeast cell surface (x axis). (c) Flow cytometry histograms of the initial Axl library and intermediate sort products compared to wild-type Axl Ig1 (gray), measuring binding to 0.5 nM Gas6 (top row) and persistent Gas6 binding after a 30-h incubation with excess competitor (bottom row). MYD1 is also included for comparison. For clarity, only the gated population of yeast expressing Axl is shown. AU, arbitrary units. (d) Binding affinities of wild-type Axl Ig1, MYD1 and Axlnb to Gas6 as determined by KinExA. (e) Binding affinities to Gas6 of every permutation of the four mutations found in MYD1. Raw KinExA data and associated error values can be found in Supplementary Figures 2 and 3.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Binding Assay, Activation Assay, Flow Cytometry, Expressing, Incubation, Comparison

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 2 | Structural basis for high-affinity binding. (a) Gas6–MYD1 co-complex showing overall architecture and 2:2 stoichiometry. (b) MYD1 Ig1 (orange) and Gas6 LG1 (gray) domains showing the location of the four mutations in MYD1 with respect to the major binding site, which lies at the interface of these two domains. (c) Analysis of the wild-type structure (PDB code 2C5D) reveals steric crowding between the side chains of T457Gas6 and V92Axl. The V92A mutation alleviates this crowding in the MYD1 co-complex and facilitates local reorganization of side chains around V92A, exemplified by R48 and Q94. This in turn creates an elongated groove on MYD1 at the binding interface that allows reorientation of T457 on Gas6. (d) Reorientation of T457 results in capping of the N terminus of helix A. The wild-type (WT, green) and MYD1 (gray) structures are overlaid for comparison. (e) Capping stabilizes helix A, as seen by B-factor analysis (Online Methods).

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Binding Assay, Mutagenesis, Comparison

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 4 | MYD1 Fc inhibits Axl activation and downstream signaling in skov3.ip cells. (a) Wild- type (WT) Axl Fc and MYD1 Fc, but not Axlnb Fc, can inhibit Gas6-mediated Axl activation in vitro. (b) Inhibition of Axl activation leads to reduced levels of phosphorylated Akt and Erk1/2 and an increase in the epithelial marker e-cadherin. For full (uncut) blots, see Supplementary Figure 11.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Activation Assay, In Vitro, Inhibition, Marker

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 5 | Sequestration of Gas6 by MYD1 Fc inhibits metastasis. (a) Amount of free Gas6 in serum of mice 12 h after administration of a single dose of MYD1 Fc. (b) Kinetics of Gas6 sequestration (black) and MYD1 Fc clearance (red) following a 1 mg per kg body weight dose of MYD1 Fc. (c) Using the off-rates of the Gas6-Axl Fc interactions (Fig. 3b), dissociation of Gas6 bound to either wild-type Axl Fc (red) or MYD1 Fc (blue) is plotted over time. The in vivo clearance of the Axl decoy receptors as measured in c is overlaid in black. Two mice were analyzed for each data point in b and c. (d–f) Tumor burden in in vivo models of metastatic human ovarian cancer. The number of visible metastases in animals treated with Axlnb Fc, wild-type Axl Fc or MYD1 Fc was counted in the skov3.ip (d) and OVCAR (f) tumor models. Representative images of mice from each treatment group in the skov3.ip model are shown, and arrows indicate disease (e). In both models, animals were administered 10 mg per kg body weight of the indicated protein twice weekly. (g) Lung metastases in the 4T1 luciferase breast cancer model, as quantified by ex vivo bioluminescent imaging. Mice received intravenous injections of the indicated treatment twice weekly. (h) Representative bioluminescent images of lungs and spleens from each treatment group; scale bar, 1 cm. Error bars represent ± s.d., n = 6–12 mice per group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: In Vivo, Luciferase, Ex Vivo, Imaging