human gas6 hgas6  (R&D Systems)

Journal: Bone Research

Article Title: Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

doi: 10.1038/s41413-026-00528-2

Figure Lengend Snippet: The γ-carboxylated protein GAS6 is expressed by osteoblasts and activates its receptors on pre-osteoclasts. a Expression analysis by qPCR of genes encoding the known γ-carboxylated proteins. Gas6 is highlighted in red. b Gene expression analysis by qPCR of the TAM receptors Axl , Mertk and Tyro3 in bone marrow derived monocytes (d0: day 0) and in differentiating osteoclast cultures in the presence of RANKL and M-CSF (d2-d6: day 2 to 6; n = 4). c Western blot analysis of the phosphorylation (P) of AKT (S473) and TAM (Y702 in AXL and Y753 in MerTK) in bone marrow derived monocytes (BMMC) serum starved for 3 h and treated with GAS6 (200 ng/mL) for the indicated times. Total AKT, MerTK, and AXL were used as loading controls. d Quantification of P-AKT (S473), P-MerTK (Y753), and P-AXL (Y702) normalized to the amount of total protein ( n = 3). Results represent the mean ± SEM

Article Snippet: GAS6 from serum and osteoblast supernatant were quantified using a mouse GAS6 ELISA (DuoSet ELISA DY986, R&D Systems, Minneapolis, MN) as we previously described.

Techniques: Expressing, Gene Expression, Derivative Assay, Western Blot, Phospho-proteomics

Journal: Bone Research

Article Title: Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

doi: 10.1038/s41413-026-00528-2

Figure Lengend Snippet: TAM signaling and γ-carboxylated GAS6 signaling promotes osteoclast formation in culture. Representative TRAP staining (left) and quantification of the TRAP + osteoclast area (right) in WT osteoblast (OB) and bone marrow cell (BM) co-cultures at day 8 in the presence of PGE 2 and VitD 3 , with or without the TAM inhibitors LDC1267 ( a ) or R428 ( b ), at the indicated concentrations. c–f Bone marrow derived monocytes (BMMC) were cultured in the presence of RANKL (20 ng/mL) and M-CSF (10 ng/mL) with or without recombinant γ-carboxylated GAS6 at the indicated concentrations for up to 6 days. c Representative TRAP staining at day 5 and 6 of differentiation. d Quantification of the TRAP + osteoclast area at day 4, 5, and 6 of differentiation. e Quantification of the number of TRAP + multinucleated osteoclasts at day 4, 5, and 6 of differentiation. f Quantification of the number of nuclei per osteoclast at day 5 of differentiation. Results represent the mean ± SEM. One-way ANOVA with Bonferroni’s posttests was used in ( a , b ) and ( d – f ). *** P < 0.001, ** P < 0.01, * P < 0.05, ns: non-significant

Article Snippet: GAS6 from serum and osteoblast supernatant were quantified using a mouse GAS6 ELISA (DuoSet ELISA DY986, R&D Systems, Minneapolis, MN) as we previously described.

Techniques: Staining, Derivative Assay, Cell Culture, Recombinant

Journal: Bone Research

Article Title: Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

doi: 10.1038/s41413-026-00528-2

Figure Lengend Snippet: Gamma-carboxylated GAS6 impacts on osteoclast differentiation and fusion. a–d Gene expression analysis by qPCR of osteoclast differentiation markers Acp5 (TRAP), Clcn7 , Ctsk , and Dcstamp . Bone marrow derived monocytes (BMMC) were cultured in the presence of RANKL (20 ng/mL) and M-CSF (10 ng/mL) with or without recombinant γ-carboxylated GAS6 at the indicated concentrations for 2, 4, and 6 days ( n = 4 per condition). e Schematic representation of the assay used to assess the impact of γ-carboxylated GAS6 on pre-osteoclast fusion in culture using a conditionally activated tdTomato (Tom) reporter (created with BioRender). f Representative pictures of live osteoclast cultures at the indicated time and concentration of recombinant γ-carboxylated GAS6. The stars indicate the presence of fusion events (Tom + cells) in presence of GAS6 at Day 4. g Quantification of the number of fusion events per 10 mm 2 at the indicated time of osteoclasts differentiation ( n = 16 fields per condition). Results represent the mean ± SEM. One-way ANOVA with Bonferroni’s posttests was used in ( a – d ) and ( g ). *** P < 0.001, ** P < 0.01

Article Snippet: GAS6 from serum and osteoblast supernatant were quantified using a mouse GAS6 ELISA (DuoSet ELISA DY986, R&D Systems, Minneapolis, MN) as we previously described.

Techniques: Gene Expression, Derivative Assay, Cell Culture, Recombinant, Concentration Assay

Journal: Bone Research

Article Title: Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

doi: 10.1038/s41413-026-00528-2

Figure Lengend Snippet: GAS6 promotes osteoclast formation and bone resorption in vivo. a–j Six-month-old WT (non-transgenic littermates) and ApoE-Gas6 Tg male mice were analyzed. GAS6 concentration in the serum ( a ) and bone marrow cavity ( b ) ( n = 4). c Representative pictures of sections from lumbar vertebrae stained with von Kossa and van Gieson. d Quantification of trabecular bone volume over tissue volume (BV/TV) from the L4 and L5 lumbar vertebrae sections ( n = 15–17). e–h μCT analysis of the distal femur trabecular bone ( n = 12). e Representative μCT images. f Quantification of trabecular bone volume (BV/TV). g Quantification of trabecular bone surface density (BS/TV). h Trabecular bone µCT derived data. Tb.Sp., Tb.N., and Tb.Th., trabecular spacing, number, and thickness, respectively; Conn.Dn., connectivity density. i–j Bone histomorphometry analysis of lumbar vertebrae in six-month-old WT and ApoE-Gas6 Tg male ( n = 19–20). i Representative pictures of TRAP staining. j Number of osteoclasts per bone perimeter (N.Oc/B.Pm) and osteoclast surface over bone surface (Oc.S/BS). Results represent the mean ± SEM. Unpaired, 2-tailed Student’s t test was used in ( a , b , d , f – h and j ). *** P < 0.001, ** P < 0.01, * P < 0.05, ns: non-significant

Article Snippet: GAS6 from serum and osteoblast supernatant were quantified using a mouse GAS6 ELISA (DuoSet ELISA DY986, R&D Systems, Minneapolis, MN) as we previously described.

Techniques: In Vivo, Transgenic Assay, Concentration Assay, Staining, Derivative Assay

Journal: Bone Research

Article Title: Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

doi: 10.1038/s41413-026-00528-2

Figure Lengend Snippet: Increased bone mass in male mice lacking γ-carboxylation in osteoblasts. a Gene expression analysis by qPCR of Ggcx and Vkorc1 in bone marrow derived monocytes (BMMC), osteoclasts (OCL), proliferating pre-osteoblasts (pre-OB), and mineralized osteoblast (OB) cultures ( n = 3). b Protein expression in liver (Liv) and bone cells by Western blot. GGCX was analyzed on a 7.5% SDS Tris Glycine gel using 20 μg of extracts, while VKORC1 was resolved on a 10% SDS Tris Tricine gel using 10 μg of extracts. c–g Six-month-old Ggcx ff and Ggcx ff ;OCN-Cre male mice were analyzed ( n = 7–10). c Representative pictures of sections from lumbar vertebrae stained with von Kossa and van Gieson. d Quantification of trabecular bone volume over tissue volume (BV/TV) from the L4 and L5 lumbar vertebrae sections. e Representative μCT images of the distal femur trabecular bones. f Quantification of trabecular bone volume (BV/TV) and trabecular bone surface density (BS/TV) from the μCT data. g Trabecular bone µCT derived data. Tb.Sp., Tb.N., and Tb.Th., trabecular spacing, number, and thickness respectively; Conn.Dn., connectivity density. Unpaired, 2-tailed Student’s t test was used in ( d ), ( f ), and ( g ). *** P < 0.001, ** P < 0.01, * P < 0.05, ns: non-significant

Article Snippet: GAS6 from serum and osteoblast supernatant were quantified using a mouse GAS6 ELISA (DuoSet ELISA DY986, R&D Systems, Minneapolis, MN) as we previously described.

Techniques: Gene Expression, Derivative Assay, Expressing, Western Blot, Staining

Journal: Bone Research

Article Title: Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

doi: 10.1038/s41413-026-00528-2

Figure Lengend Snippet: Reduced osteoclast number and surface in Ggcx ff ;OCN-Cre male mice. a–h Bone histomorphometry analysis of lumbar vertebrae in six-month-old Ggcx ff and Ggcx ff ;OCN-Cre male mice ( n = 6–10). a Representative pictures of calcein double labeling and toluidine blue staining. b Mineral apposition rate (MAR). c Bone formation rate over bone surface (BFR/BS). d Number of osteoblasts per bone perimeter (N.Ob/B.Pm). e Osteoblast surface over bone surface (Ob.S/BS). f Representative pictures of TRAP staining. g Number of osteoclasts per bone perimeter (N.Oc/B.Pm). h Osteoclast surface over bone surface (Oc.S/BS). i Fasting serum CTx levels ( n = 12–17). Results represent the mean ± SEM. Unpaired, 2-tailed Student’s t test was used in ( b – e ) and ( g – i ). ** P < 0.01, * P < 0.05, ns: non-significant

Article Snippet: GAS6 from serum and osteoblast supernatant were quantified using a mouse GAS6 ELISA (DuoSet ELISA DY986, R&D Systems, Minneapolis, MN) as we previously described.

Techniques: Labeling, Staining

Journal: Bone Research

Article Title: Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

doi: 10.1038/s41413-026-00528-2

Figure Lengend Snippet: Ggcx inactivation impairs the ability of osteoblasts to support osteoclastogenesis ex vivo. a Representative TRAP staining of osteoblasts (OB) and bone marrow cells (BM) co-cultures at day 8 in the presence of prostaglandin E 2 (PGE 2 ; 10 –6 mol/L) and 1,25 vitamin D 3 (VitD 3 ; 10 –8 mol/L). Ggcx ff osteoblasts were transduced with either Ad-GFP (control) or Ad-Cre (knockout) before the addition of the WT bone marrow cells. b Quantification of the number of TRAP + osteoclasts per well (Nb of OCL/well) ( n = 3). c Representative TRAP staining of osteoblasts and bone marrow cells co-cultures at day 8. Control ( Ocn + / + ) or osteocalcin-deficient ( Ocn -/- ) osteoblasts were cultured with WT bone marrow cells. d Quantification of the number of TRAP + osteoclasts per well (Nb of OCL/well) ( n = 3). e Gene expression analysis by qPCR in Ggcx ff + Ad-GFP and Ggcx ff + Ad-Cre osteoblasts cultured in presence (+) or absence (–) of PGE 2 and VitD 3 for 6 days. Results represent the mean ± SEM. Unpaired, 2-tailed Student’s t test was used in ( b ) and ( d ). Two-way ANOVA with Bonferroni’s posttests was used in ( e ). *** P < 0.001, ** P < 0.01, * P < 0.05, ns: non-significant

Article Snippet: GAS6 from serum and osteoblast supernatant were quantified using a mouse GAS6 ELISA (DuoSet ELISA DY986, R&D Systems, Minneapolis, MN) as we previously described.

Techniques: Ex Vivo, Staining, Transduction, Control, Knock-Out, Cell Culture, Gene Expression

Journal: Bone Research

Article Title: Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

doi: 10.1038/s41413-026-00528-2

Figure Lengend Snippet: The γ-carboxylated protein GAS6 is expressed by osteoblasts and activates its receptors on pre-osteoclasts. a Expression analysis by qPCR of genes encoding the known γ-carboxylated proteins. Gas6 is highlighted in red. b Gene expression analysis by qPCR of the TAM receptors Axl , Mertk and Tyro3 in bone marrow derived monocytes (d0: day 0) and in differentiating osteoclast cultures in the presence of RANKL and M-CSF (d2-d6: day 2 to 6; n = 4). c Western blot analysis of the phosphorylation (P) of AKT (S473) and TAM (Y702 in AXL and Y753 in MerTK) in bone marrow derived monocytes (BMMC) serum starved for 3 h and treated with GAS6 (200 ng/mL) for the indicated times. Total AKT, MerTK, and AXL were used as loading controls. d Quantification of P-AKT (S473), P-MerTK (Y753), and P-AXL (Y702) normalized to the amount of total protein ( n = 3). Results represent the mean ± SEM

Article Snippet: GAS6 from serum and osteoblast supernatant were quantified using a mouse GAS6 ELISA (DuoSet ELISA DY986, R&D Systems, Minneapolis, MN) as we previously described.

Techniques: Expressing, Gene Expression, Derivative Assay, Western Blot, Phospho-proteomics

Journal: Bone Research

Article Title: Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

doi: 10.1038/s41413-026-00528-2

Figure Lengend Snippet: TAM signaling and γ-carboxylated GAS6 signaling promotes osteoclast formation in culture. Representative TRAP staining (left) and quantification of the TRAP + osteoclast area (right) in WT osteoblast (OB) and bone marrow cell (BM) co-cultures at day 8 in the presence of PGE 2 and VitD 3 , with or without the TAM inhibitors LDC1267 ( a ) or R428 ( b ), at the indicated concentrations. c–f Bone marrow derived monocytes (BMMC) were cultured in the presence of RANKL (20 ng/mL) and M-CSF (10 ng/mL) with or without recombinant γ-carboxylated GAS6 at the indicated concentrations for up to 6 days. c Representative TRAP staining at day 5 and 6 of differentiation. d Quantification of the TRAP + osteoclast area at day 4, 5, and 6 of differentiation. e Quantification of the number of TRAP + multinucleated osteoclasts at day 4, 5, and 6 of differentiation. f Quantification of the number of nuclei per osteoclast at day 5 of differentiation. Results represent the mean ± SEM. One-way ANOVA with Bonferroni’s posttests was used in ( a , b ) and ( d – f ). *** P < 0.001, ** P < 0.01, * P < 0.05, ns: non-significant

Article Snippet: GAS6 from serum and osteoblast supernatant were quantified using a mouse GAS6 ELISA (DuoSet ELISA DY986, R&D Systems, Minneapolis, MN) as we previously described.

Techniques: Staining, Derivative Assay, Cell Culture, Recombinant

Journal: NPJ Precision Oncology

Article Title: AXL–SHC1 signaling axis mediates adaptive resistance to HER2-targeted tyrosine kinase inhibitors in HER2-aberrant lung and gastric cancers

doi: 10.1038/s41698-026-01385-2

Figure Lengend Snippet: A qPCR analysis of GAS6 expression in Calu-3 and MKN7 parent cells treated with nonspecific (control) or GAS6-specific siRNAs for 48 h. * P < 0.05 (unpaired t-tests). B MTT assays evaluating cell viability of Calu-3 and MKN7 cells treated with nonspecific or GAS6-specific siRNAs were incubated with or without mobocertinib (0.01 or 0.1 μmol/L) for 72 h. These cell lines were incubated in an RPMI 1640 medium with 0.1% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) in a humidified 5% CO2 incubator at 37 °C for 72 h (* P < 0.05, one-way ANOVA). C Western blotting analysis of Calu-3 and MKN7 cells transfected with nonspecific or GAS6-specific siRNAs for 48 h, followed by treatment with or without mobocertinib (0.01 or 0.1 μmol/L) for 4 h. D Calu-3 and MKN7 cells with nonspecific siRNA or AXL-specific siRNAs were treated for 72 h with or without mobocertinib (0.01 or 0.1 μmol/L) and/or GAS6 (50 ng/mL). Cell growth was determined using MTT assays. * P < 0.05 (two-way ANOVA). E Western blotting of Calu-3 and MKN7 cells transfected with nonspecific or AXL-specific siRNAs and treated with or without mobocertinib (0.01 or 0.1 μmol/L) for 4 h and GAS6 (50 ng/mL) for 15 min. F MKN7 cells treated with or without mobocertinib (Mobo, 0.1 μmol/L) for 24 h were detected by western blotting with immunoprecipitation of the indicated proteins.

Article Snippet: Recombinant human GAS6 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Control, Incubation, Western Blot, Transfection, Immunoprecipitation

Journal: NPJ Precision Oncology

Article Title: AXL–SHC1 signaling axis mediates adaptive resistance to HER2-targeted tyrosine kinase inhibitors in HER2-aberrant lung and gastric cancers

doi: 10.1038/s41698-026-01385-2

Figure Lengend Snippet: A Combination of mobocertinib and knockdown of several adapter proteins in Calu-3. and MKN7 cells. Calu-3 and MKN7 cells treated with nonspecific control, FYN, GRB2, PTPN11, Shc1, or SRC-specific siRNAs were incubated with or without mobocertinib (0.01 or 0.1 μmol/L) for 72 h, and cell viability was detected using MTT assays. * P < 0.05 compared with nonspecific control siRNA (two-way ANOVA). Data are represented as the mean ± S.D. B Western blotting of Calu-3 and MKN7 cells treated with nonspecific control siRNA or Shc1-specific siRNA and incubated with or without mobocertinib (0.01 or 0.1 μmol/L) for 24 h. C Calu-3 and MKN7 cells treated with or without mobocertinib (Mobo; 0.01 or 0.1 μmol/L) for 24 h were detected by western blotting with immunoprecipitation of the indicated proteins. D Western blotting showing nuclear localization of ShcBP1 in Calu-3 and MKN7 cells treated with mobocertinib (0.01 or 0.1 μmol/L) for 72 h. E Immunofluorescence showing nuclear localization of ShcBP1 in Calu-3 and MKN7 cells treated with mobocertinib (0.01 or 0.1 μmol/L) for 72 h. F MTT assays assessing cell viability of Calu-3 and MKN7 treated with nonspecific siRNA or ShcBP1-specific siRNAs, and incubated with or without mobocertinib * P < 0.05 (one-way ANOVA). G Schematic diagram illustrating the drug tolerance mechanisms, including GAS6–AXL activation through the SHC1BP–SHC1 complex, in HER2-aberrant cancer cells.

Article Snippet: Recombinant human GAS6 was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Knockdown, Control, Incubation, Western Blot, Immunoprecipitation, Immunofluorescence, Activation Assay