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gas6 standard  (R&D Systems)


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    R&D Systems gas6 standard
    Figure 1. Differentiation of THP-1 cells induced by PMA. (A) THP-1 monocytes are differentiated to macrophages and induced to express Mertk by the addition of 100 ng/mL of PMA for at least 24 h, while Axl expression is not induced. Likewise, Tyro3 expression remains stable and is unaffected by treatment. H1299 and Beas2B cells are used as positive controls for Mertk/Axl and Tyro3, respec- tively. The observed doublet for Mertk corresponds to fully glycosylated and partially glycosylated glycoforms. (B) THP-1 cells treated with 100 ng/mL of PMA over 48 h exhibit a decreased expression of <t>Gas6</t> but an increased expression of Protein S. (C) MertK and Gas6 expression patterns over a time of 48 h after 100 ng/mL of PMA treatment as quantified from Western blots. (D) Bar plots showing MerTK and Gas6 expression quantified from Western blots at 0 h and post 72 h of 100 ng/mL of PMA treatment (significant differences were observed between the 0 h and 72 h time points for Gas6 (* p = 0.029) and MerTK (** p = 0.003), as determined by independent t-tests). (E) THP-1 cells were treated with 100 ng/mL of PMA over 72 h. mRNA was analyzed via qRT-PCR for Mertk, Axl, Protein S (ProS), Tyro3, and Gas6. Mertk transcription was highly elevated, more than 10-fold, due to PMA treatment, while Gas6 transcription was repressed more than 10-fold. (F) PMA treatment of THP-1s illustrates a complimentary phenomenon. As a monocyte, Gas6 is expressed with little Mertk expression; however, when differentiated, Mertk is highly expressed in favor of Gas6.
    Gas6 Standard, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gas6 standard/product/R&D Systems
    Average 95 stars, based on 85 article reviews
    gas6 standard - by Bioz Stars, 2026-03
    95/100 stars

    Images

    1) Product Images from "Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing."

    Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

    Journal: International journal of molecular sciences

    doi: 10.3390/ijms25084404

    Figure 1. Differentiation of THP-1 cells induced by PMA. (A) THP-1 monocytes are differentiated to macrophages and induced to express Mertk by the addition of 100 ng/mL of PMA for at least 24 h, while Axl expression is not induced. Likewise, Tyro3 expression remains stable and is unaffected by treatment. H1299 and Beas2B cells are used as positive controls for Mertk/Axl and Tyro3, respec- tively. The observed doublet for Mertk corresponds to fully glycosylated and partially glycosylated glycoforms. (B) THP-1 cells treated with 100 ng/mL of PMA over 48 h exhibit a decreased expression of Gas6 but an increased expression of Protein S. (C) MertK and Gas6 expression patterns over a time of 48 h after 100 ng/mL of PMA treatment as quantified from Western blots. (D) Bar plots showing MerTK and Gas6 expression quantified from Western blots at 0 h and post 72 h of 100 ng/mL of PMA treatment (significant differences were observed between the 0 h and 72 h time points for Gas6 (* p = 0.029) and MerTK (** p = 0.003), as determined by independent t-tests). (E) THP-1 cells were treated with 100 ng/mL of PMA over 72 h. mRNA was analyzed via qRT-PCR for Mertk, Axl, Protein S (ProS), Tyro3, and Gas6. Mertk transcription was highly elevated, more than 10-fold, due to PMA treatment, while Gas6 transcription was repressed more than 10-fold. (F) PMA treatment of THP-1s illustrates a complimentary phenomenon. As a monocyte, Gas6 is expressed with little Mertk expression; however, when differentiated, Mertk is highly expressed in favor of Gas6.
    Figure Legend Snippet: Figure 1. Differentiation of THP-1 cells induced by PMA. (A) THP-1 monocytes are differentiated to macrophages and induced to express Mertk by the addition of 100 ng/mL of PMA for at least 24 h, while Axl expression is not induced. Likewise, Tyro3 expression remains stable and is unaffected by treatment. H1299 and Beas2B cells are used as positive controls for Mertk/Axl and Tyro3, respec- tively. The observed doublet for Mertk corresponds to fully glycosylated and partially glycosylated glycoforms. (B) THP-1 cells treated with 100 ng/mL of PMA over 48 h exhibit a decreased expression of Gas6 but an increased expression of Protein S. (C) MertK and Gas6 expression patterns over a time of 48 h after 100 ng/mL of PMA treatment as quantified from Western blots. (D) Bar plots showing MerTK and Gas6 expression quantified from Western blots at 0 h and post 72 h of 100 ng/mL of PMA treatment (significant differences were observed between the 0 h and 72 h time points for Gas6 (* p = 0.029) and MerTK (** p = 0.003), as determined by independent t-tests). (E) THP-1 cells were treated with 100 ng/mL of PMA over 72 h. mRNA was analyzed via qRT-PCR for Mertk, Axl, Protein S (ProS), Tyro3, and Gas6. Mertk transcription was highly elevated, more than 10-fold, due to PMA treatment, while Gas6 transcription was repressed more than 10-fold. (F) PMA treatment of THP-1s illustrates a complimentary phenomenon. As a monocyte, Gas6 is expressed with little Mertk expression; however, when differentiated, Mertk is highly expressed in favor of Gas6.

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

    Figure 3. γ-carboxylated Gas6 Reduces Full-length Mertk Expression and Decreases sMertk. (A) γ- carboxylation status (Gla) of Gas6 is regulated with the addition of Vitamin K or warfarin, producing either a γ-carboxylated, active ligand or a non-γ-carboxylated, inactive ligand, respectively. The carboxylation status (Gla) domain of Gas6 can bind with externalized PS. (B) Recombinant inactive (Gas6-W) and active (Gas6-VK) were produced via HEK293 transfection, with a mock transfection control (Mock). The amount of Gas6 was observed (bottom), while the carboxylation status that is responsible for ligand activity was determined for each (left, top). (C) PMA-differentiated THP-1s were treated for 4 h with either serum-free RPMI only (-), a mock transfection control, 10 nM of active Gas6 (Gas6-VK), or 10 nM of inactive Gas6 (Gas6-W). Treatments were alone or combined with inhibitors of 3 µM of GW280264X (an ADAM17 inhibitor), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a proteasomal inhibitor). (D) Quantitative results indicate that Gas6, either active or inactive, does not stabilize the C-terminal fragments as shown in the DAPT and MG132 treatments (bands at 75 kDa). As denoted by sMertk (top), the cleavage is decreased with GW280264X treatment compared to the untreated control.
    Figure Legend Snippet: Figure 3. γ-carboxylated Gas6 Reduces Full-length Mertk Expression and Decreases sMertk. (A) γ- carboxylation status (Gla) of Gas6 is regulated with the addition of Vitamin K or warfarin, producing either a γ-carboxylated, active ligand or a non-γ-carboxylated, inactive ligand, respectively. The carboxylation status (Gla) domain of Gas6 can bind with externalized PS. (B) Recombinant inactive (Gas6-W) and active (Gas6-VK) were produced via HEK293 transfection, with a mock transfection control (Mock). The amount of Gas6 was observed (bottom), while the carboxylation status that is responsible for ligand activity was determined for each (left, top). (C) PMA-differentiated THP-1s were treated for 4 h with either serum-free RPMI only (-), a mock transfection control, 10 nM of active Gas6 (Gas6-VK), or 10 nM of inactive Gas6 (Gas6-W). Treatments were alone or combined with inhibitors of 3 µM of GW280264X (an ADAM17 inhibitor), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a proteasomal inhibitor). (D) Quantitative results indicate that Gas6, either active or inactive, does not stabilize the C-terminal fragments as shown in the DAPT and MG132 treatments (bands at 75 kDa). As denoted by sMertk (top), the cleavage is decreased with GW280264X treatment compared to the untreated control.

    Techniques Used: Expressing, Recombinant, Produced, Transfection, Control, Activity Assay

    Figure 5. γ-carboxylated Gas6 reduces the tagged Mertk construct on cell membranes of THP-1 cells. (A) THP-1s expressing the tagged construct were starved for 18 h in serum-free RPMI and treated for 3 h. Flow cytometry data shows positive GFPs without staining. As expected, CHX treatment after 3 h reduces the expression of the construct. Gas6-VK, used at a concentration > 10 nM, decreases the FLAG-PE signal more when compared to other treatments known to induce cleavage (PMA, LPS). GW280264X is used as a control for Mertk cleavage. (B) Histogram analysis of “A.” Gas6-VK induces a shift in the FLAG-PE signal, showing that less surface Mertk is present. γ-Carboxylated Gas6 induced the degradation of Mertk on the cell membrane. (C) THP-1s treated with γ-Carboxylated Gas6 at a 10 nM concentration show increased MerTK phosphorylation, detected by immunoblotting against pMerTK. (D) THP-1s expressing the tagged construct, treated with >10 nM of Gas6-VK + 1 µM of PS, show a reduced level of both domains of the tagged construct, suggesting that the Gas6-VK + PS treatment is leading to a degradation of the full-length receptor.
    Figure Legend Snippet: Figure 5. γ-carboxylated Gas6 reduces the tagged Mertk construct on cell membranes of THP-1 cells. (A) THP-1s expressing the tagged construct were starved for 18 h in serum-free RPMI and treated for 3 h. Flow cytometry data shows positive GFPs without staining. As expected, CHX treatment after 3 h reduces the expression of the construct. Gas6-VK, used at a concentration > 10 nM, decreases the FLAG-PE signal more when compared to other treatments known to induce cleavage (PMA, LPS). GW280264X is used as a control for Mertk cleavage. (B) Histogram analysis of “A.” Gas6-VK induces a shift in the FLAG-PE signal, showing that less surface Mertk is present. γ-Carboxylated Gas6 induced the degradation of Mertk on the cell membrane. (C) THP-1s treated with γ-Carboxylated Gas6 at a 10 nM concentration show increased MerTK phosphorylation, detected by immunoblotting against pMerTK. (D) THP-1s expressing the tagged construct, treated with >10 nM of Gas6-VK + 1 µM of PS, show a reduced level of both domains of the tagged construct, suggesting that the Gas6-VK + PS treatment is leading to a degradation of the full-length receptor.

    Techniques Used: Construct, Expressing, Flow Cytometry, Staining, Concentration Assay, Control, Membrane, Phospho-proteomics, Western Blot

    Figure 6. Confocal imaging of the GFP-tagged MerTK construct displayed the differential localization of MerTK upon ligand stimulation and the inhibition of proteases. (A) DAPT and MG132 treatments induce increased cytoplasmic GFP signals compared to untreated cells. (B) Confocal imaging shows GFP localized on the cell membrane, indicating the presence of tagged Mertk constructs on the cell surface. γ-Carboxylated Gas6-treated tagged THP-1 cells showed a reduction in the MerTK construct from the membrane and the localization in lysosomes. (C) Quantification of the GFP fluorescence intensity per cell, calculated from confocal images in mock, γ-carboxylated Gas6, and non-γ-carboxylated Gas6, with the bar plots showing the mean and standard error of each treatment. ns; non-significant; *** p < 0.001; **** p < 0.0001.
    Figure Legend Snippet: Figure 6. Confocal imaging of the GFP-tagged MerTK construct displayed the differential localization of MerTK upon ligand stimulation and the inhibition of proteases. (A) DAPT and MG132 treatments induce increased cytoplasmic GFP signals compared to untreated cells. (B) Confocal imaging shows GFP localized on the cell membrane, indicating the presence of tagged Mertk constructs on the cell surface. γ-Carboxylated Gas6-treated tagged THP-1 cells showed a reduction in the MerTK construct from the membrane and the localization in lysosomes. (C) Quantification of the GFP fluorescence intensity per cell, calculated from confocal images in mock, γ-carboxylated Gas6, and non-γ-carboxylated Gas6, with the bar plots showing the mean and standard error of each treatment. ns; non-significant; *** p < 0.001; **** p < 0.0001.

    Techniques Used: Imaging, Construct, Inhibition, Membrane, Fluorescence

    Figure 7. γ-carboxylation of Gas6 induces Mertk degradation independent of phosphorylation. (A) Mutants of the tagged construct were created. K619M, a substitution in the ATP-binding site (underlined) of the Mertk kinase domain, inhibits autophosphorylation by preventing ATP binding and the exchange of phosphate molecules. * Amino acid position for ADAM17 cleavage. (B) Constructs are treated with 10 nM ofGas6-VK for 30 min after a 6 h serum starvation. With the ability to bind ATP and phosphorylate, the WT construct becomes phosphorylated while the kinase dead K619M mutant does not. (C) Mutant constructs were treated with 3 µM of GW280264X (an ADAM17 inhibitor; GW), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a protea- somal inhibitor) for 4 h. Results show that the absence of the K619M C-terminal fragment from the MG132 treatment with the addition of GW indicates the fragment is a product of cleavage. (D) Confocal images indicated that the K619M mutants have more cytoplasmatic c-Mertk than WT Mertk with MG132 treatment. (E) Comparison of the contrasting pathways between Notch Recep- tor and MerTK Signaling Models. In the absence of a ligand, Notch is not cleaved, while MerTK undergoes homeostatic cleavage, leading to proteasomal degradation. Upon ligand binding, Notch is cleaved at the ADAM 17 site, revealing the gamma-secretase site. Subsequent gamma-secretase cleavage releases the Notch intracellular domain, translocating it to the nucleus for transcriptional activation. Conversely, MerTK, upon ligand (Gas6) interaction, is internalized into endosomal compartments and localizes within lysosomes.
    Figure Legend Snippet: Figure 7. γ-carboxylation of Gas6 induces Mertk degradation independent of phosphorylation. (A) Mutants of the tagged construct were created. K619M, a substitution in the ATP-binding site (underlined) of the Mertk kinase domain, inhibits autophosphorylation by preventing ATP binding and the exchange of phosphate molecules. * Amino acid position for ADAM17 cleavage. (B) Constructs are treated with 10 nM ofGas6-VK for 30 min after a 6 h serum starvation. With the ability to bind ATP and phosphorylate, the WT construct becomes phosphorylated while the kinase dead K619M mutant does not. (C) Mutant constructs were treated with 3 µM of GW280264X (an ADAM17 inhibitor; GW), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a protea- somal inhibitor) for 4 h. Results show that the absence of the K619M C-terminal fragment from the MG132 treatment with the addition of GW indicates the fragment is a product of cleavage. (D) Confocal images indicated that the K619M mutants have more cytoplasmatic c-Mertk than WT Mertk with MG132 treatment. (E) Comparison of the contrasting pathways between Notch Recep- tor and MerTK Signaling Models. In the absence of a ligand, Notch is not cleaved, while MerTK undergoes homeostatic cleavage, leading to proteasomal degradation. Upon ligand binding, Notch is cleaved at the ADAM 17 site, revealing the gamma-secretase site. Subsequent gamma-secretase cleavage releases the Notch intracellular domain, translocating it to the nucleus for transcriptional activation. Conversely, MerTK, upon ligand (Gas6) interaction, is internalized into endosomal compartments and localizes within lysosomes.

    Techniques Used: Phospho-proteomics, Construct, Binding Assay, Mutagenesis, Comparison, Ligand Binding Assay, Activation Assay



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    Figure 1. Differentiation of THP-1 cells induced by PMA. (A) THP-1 monocytes are differentiated to macrophages and induced to express Mertk by the addition of 100 ng/mL of PMA for at least 24 h, while Axl expression is not induced. Likewise, Tyro3 expression remains stable and is unaffected by treatment. H1299 and Beas2B cells are used as positive controls for Mertk/Axl and Tyro3, respec- tively. The observed doublet for Mertk corresponds to fully glycosylated and partially glycosylated glycoforms. (B) THP-1 cells treated with 100 ng/mL of PMA over 48 h exhibit a decreased expression of <t>Gas6</t> but an increased expression of Protein S. (C) MertK and Gas6 expression patterns over a time of 48 h after 100 ng/mL of PMA treatment as quantified from Western blots. (D) Bar plots showing MerTK and Gas6 expression quantified from Western blots at 0 h and post 72 h of 100 ng/mL of PMA treatment (significant differences were observed between the 0 h and 72 h time points for Gas6 (* p = 0.029) and MerTK (** p = 0.003), as determined by independent t-tests). (E) THP-1 cells were treated with 100 ng/mL of PMA over 72 h. mRNA was analyzed via qRT-PCR for Mertk, Axl, Protein S (ProS), Tyro3, and Gas6. Mertk transcription was highly elevated, more than 10-fold, due to PMA treatment, while Gas6 transcription was repressed more than 10-fold. (F) PMA treatment of THP-1s illustrates a complimentary phenomenon. As a monocyte, Gas6 is expressed with little Mertk expression; however, when differentiated, Mertk is highly expressed in favor of Gas6.
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    Figure 1. Differentiation of THP-1 cells induced by PMA. (A) THP-1 monocytes are differentiated to macrophages and induced to express Mertk by the addition of 100 ng/mL of PMA for at least 24 h, while Axl expression is not induced. Likewise, Tyro3 expression remains stable and is unaffected by treatment. H1299 and Beas2B cells are used as positive controls for Mertk/Axl and Tyro3, respec- tively. The observed doublet for Mertk corresponds to fully glycosylated and partially glycosylated glycoforms. (B) THP-1 cells treated with 100 ng/mL of PMA over 48 h exhibit a decreased expression of Gas6 but an increased expression of Protein S. (C) MertK and Gas6 expression patterns over a time of 48 h after 100 ng/mL of PMA treatment as quantified from Western blots. (D) Bar plots showing MerTK and Gas6 expression quantified from Western blots at 0 h and post 72 h of 100 ng/mL of PMA treatment (significant differences were observed between the 0 h and 72 h time points for Gas6 (* p = 0.029) and MerTK (** p = 0.003), as determined by independent t-tests). (E) THP-1 cells were treated with 100 ng/mL of PMA over 72 h. mRNA was analyzed via qRT-PCR for Mertk, Axl, Protein S (ProS), Tyro3, and Gas6. Mertk transcription was highly elevated, more than 10-fold, due to PMA treatment, while Gas6 transcription was repressed more than 10-fold. (F) PMA treatment of THP-1s illustrates a complimentary phenomenon. As a monocyte, Gas6 is expressed with little Mertk expression; however, when differentiated, Mertk is highly expressed in favor of Gas6.

    Journal: International journal of molecular sciences

    Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

    doi: 10.3390/ijms25084404

    Figure Lengend Snippet: Figure 1. Differentiation of THP-1 cells induced by PMA. (A) THP-1 monocytes are differentiated to macrophages and induced to express Mertk by the addition of 100 ng/mL of PMA for at least 24 h, while Axl expression is not induced. Likewise, Tyro3 expression remains stable and is unaffected by treatment. H1299 and Beas2B cells are used as positive controls for Mertk/Axl and Tyro3, respec- tively. The observed doublet for Mertk corresponds to fully glycosylated and partially glycosylated glycoforms. (B) THP-1 cells treated with 100 ng/mL of PMA over 48 h exhibit a decreased expression of Gas6 but an increased expression of Protein S. (C) MertK and Gas6 expression patterns over a time of 48 h after 100 ng/mL of PMA treatment as quantified from Western blots. (D) Bar plots showing MerTK and Gas6 expression quantified from Western blots at 0 h and post 72 h of 100 ng/mL of PMA treatment (significant differences were observed between the 0 h and 72 h time points for Gas6 (* p = 0.029) and MerTK (** p = 0.003), as determined by independent t-tests). (E) THP-1 cells were treated with 100 ng/mL of PMA over 72 h. mRNA was analyzed via qRT-PCR for Mertk, Axl, Protein S (ProS), Tyro3, and Gas6. Mertk transcription was highly elevated, more than 10-fold, due to PMA treatment, while Gas6 transcription was repressed more than 10-fold. (F) PMA treatment of THP-1s illustrates a complimentary phenomenon. As a monocyte, Gas6 is expressed with little Mertk expression; however, when differentiated, Mertk is highly expressed in favor of Gas6.

    Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    Figure 3. γ-carboxylated Gas6 Reduces Full-length Mertk Expression and Decreases sMertk. (A) γ- carboxylation status (Gla) of Gas6 is regulated with the addition of Vitamin K or warfarin, producing either a γ-carboxylated, active ligand or a non-γ-carboxylated, inactive ligand, respectively. The carboxylation status (Gla) domain of Gas6 can bind with externalized PS. (B) Recombinant inactive (Gas6-W) and active (Gas6-VK) were produced via HEK293 transfection, with a mock transfection control (Mock). The amount of Gas6 was observed (bottom), while the carboxylation status that is responsible for ligand activity was determined for each (left, top). (C) PMA-differentiated THP-1s were treated for 4 h with either serum-free RPMI only (-), a mock transfection control, 10 nM of active Gas6 (Gas6-VK), or 10 nM of inactive Gas6 (Gas6-W). Treatments were alone or combined with inhibitors of 3 µM of GW280264X (an ADAM17 inhibitor), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a proteasomal inhibitor). (D) Quantitative results indicate that Gas6, either active or inactive, does not stabilize the C-terminal fragments as shown in the DAPT and MG132 treatments (bands at 75 kDa). As denoted by sMertk (top), the cleavage is decreased with GW280264X treatment compared to the untreated control.

    Journal: International journal of molecular sciences

    Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

    doi: 10.3390/ijms25084404

    Figure Lengend Snippet: Figure 3. γ-carboxylated Gas6 Reduces Full-length Mertk Expression and Decreases sMertk. (A) γ- carboxylation status (Gla) of Gas6 is regulated with the addition of Vitamin K or warfarin, producing either a γ-carboxylated, active ligand or a non-γ-carboxylated, inactive ligand, respectively. The carboxylation status (Gla) domain of Gas6 can bind with externalized PS. (B) Recombinant inactive (Gas6-W) and active (Gas6-VK) were produced via HEK293 transfection, with a mock transfection control (Mock). The amount of Gas6 was observed (bottom), while the carboxylation status that is responsible for ligand activity was determined for each (left, top). (C) PMA-differentiated THP-1s were treated for 4 h with either serum-free RPMI only (-), a mock transfection control, 10 nM of active Gas6 (Gas6-VK), or 10 nM of inactive Gas6 (Gas6-W). Treatments were alone or combined with inhibitors of 3 µM of GW280264X (an ADAM17 inhibitor), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a proteasomal inhibitor). (D) Quantitative results indicate that Gas6, either active or inactive, does not stabilize the C-terminal fragments as shown in the DAPT and MG132 treatments (bands at 75 kDa). As denoted by sMertk (top), the cleavage is decreased with GW280264X treatment compared to the untreated control.

    Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

    Techniques: Expressing, Recombinant, Produced, Transfection, Control, Activity Assay

    Figure 5. γ-carboxylated Gas6 reduces the tagged Mertk construct on cell membranes of THP-1 cells. (A) THP-1s expressing the tagged construct were starved for 18 h in serum-free RPMI and treated for 3 h. Flow cytometry data shows positive GFPs without staining. As expected, CHX treatment after 3 h reduces the expression of the construct. Gas6-VK, used at a concentration > 10 nM, decreases the FLAG-PE signal more when compared to other treatments known to induce cleavage (PMA, LPS). GW280264X is used as a control for Mertk cleavage. (B) Histogram analysis of “A.” Gas6-VK induces a shift in the FLAG-PE signal, showing that less surface Mertk is present. γ-Carboxylated Gas6 induced the degradation of Mertk on the cell membrane. (C) THP-1s treated with γ-Carboxylated Gas6 at a 10 nM concentration show increased MerTK phosphorylation, detected by immunoblotting against pMerTK. (D) THP-1s expressing the tagged construct, treated with >10 nM of Gas6-VK + 1 µM of PS, show a reduced level of both domains of the tagged construct, suggesting that the Gas6-VK + PS treatment is leading to a degradation of the full-length receptor.

    Journal: International journal of molecular sciences

    Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

    doi: 10.3390/ijms25084404

    Figure Lengend Snippet: Figure 5. γ-carboxylated Gas6 reduces the tagged Mertk construct on cell membranes of THP-1 cells. (A) THP-1s expressing the tagged construct were starved for 18 h in serum-free RPMI and treated for 3 h. Flow cytometry data shows positive GFPs without staining. As expected, CHX treatment after 3 h reduces the expression of the construct. Gas6-VK, used at a concentration > 10 nM, decreases the FLAG-PE signal more when compared to other treatments known to induce cleavage (PMA, LPS). GW280264X is used as a control for Mertk cleavage. (B) Histogram analysis of “A.” Gas6-VK induces a shift in the FLAG-PE signal, showing that less surface Mertk is present. γ-Carboxylated Gas6 induced the degradation of Mertk on the cell membrane. (C) THP-1s treated with γ-Carboxylated Gas6 at a 10 nM concentration show increased MerTK phosphorylation, detected by immunoblotting against pMerTK. (D) THP-1s expressing the tagged construct, treated with >10 nM of Gas6-VK + 1 µM of PS, show a reduced level of both domains of the tagged construct, suggesting that the Gas6-VK + PS treatment is leading to a degradation of the full-length receptor.

    Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

    Techniques: Construct, Expressing, Flow Cytometry, Staining, Concentration Assay, Control, Membrane, Phospho-proteomics, Western Blot

    Figure 6. Confocal imaging of the GFP-tagged MerTK construct displayed the differential localization of MerTK upon ligand stimulation and the inhibition of proteases. (A) DAPT and MG132 treatments induce increased cytoplasmic GFP signals compared to untreated cells. (B) Confocal imaging shows GFP localized on the cell membrane, indicating the presence of tagged Mertk constructs on the cell surface. γ-Carboxylated Gas6-treated tagged THP-1 cells showed a reduction in the MerTK construct from the membrane and the localization in lysosomes. (C) Quantification of the GFP fluorescence intensity per cell, calculated from confocal images in mock, γ-carboxylated Gas6, and non-γ-carboxylated Gas6, with the bar plots showing the mean and standard error of each treatment. ns; non-significant; *** p < 0.001; **** p < 0.0001.

    Journal: International journal of molecular sciences

    Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

    doi: 10.3390/ijms25084404

    Figure Lengend Snippet: Figure 6. Confocal imaging of the GFP-tagged MerTK construct displayed the differential localization of MerTK upon ligand stimulation and the inhibition of proteases. (A) DAPT and MG132 treatments induce increased cytoplasmic GFP signals compared to untreated cells. (B) Confocal imaging shows GFP localized on the cell membrane, indicating the presence of tagged Mertk constructs on the cell surface. γ-Carboxylated Gas6-treated tagged THP-1 cells showed a reduction in the MerTK construct from the membrane and the localization in lysosomes. (C) Quantification of the GFP fluorescence intensity per cell, calculated from confocal images in mock, γ-carboxylated Gas6, and non-γ-carboxylated Gas6, with the bar plots showing the mean and standard error of each treatment. ns; non-significant; *** p < 0.001; **** p < 0.0001.

    Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

    Techniques: Imaging, Construct, Inhibition, Membrane, Fluorescence

    Figure 7. γ-carboxylation of Gas6 induces Mertk degradation independent of phosphorylation. (A) Mutants of the tagged construct were created. K619M, a substitution in the ATP-binding site (underlined) of the Mertk kinase domain, inhibits autophosphorylation by preventing ATP binding and the exchange of phosphate molecules. * Amino acid position for ADAM17 cleavage. (B) Constructs are treated with 10 nM ofGas6-VK for 30 min after a 6 h serum starvation. With the ability to bind ATP and phosphorylate, the WT construct becomes phosphorylated while the kinase dead K619M mutant does not. (C) Mutant constructs were treated with 3 µM of GW280264X (an ADAM17 inhibitor; GW), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a protea- somal inhibitor) for 4 h. Results show that the absence of the K619M C-terminal fragment from the MG132 treatment with the addition of GW indicates the fragment is a product of cleavage. (D) Confocal images indicated that the K619M mutants have more cytoplasmatic c-Mertk than WT Mertk with MG132 treatment. (E) Comparison of the contrasting pathways between Notch Recep- tor and MerTK Signaling Models. In the absence of a ligand, Notch is not cleaved, while MerTK undergoes homeostatic cleavage, leading to proteasomal degradation. Upon ligand binding, Notch is cleaved at the ADAM 17 site, revealing the gamma-secretase site. Subsequent gamma-secretase cleavage releases the Notch intracellular domain, translocating it to the nucleus for transcriptional activation. Conversely, MerTK, upon ligand (Gas6) interaction, is internalized into endosomal compartments and localizes within lysosomes.

    Journal: International journal of molecular sciences

    Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

    doi: 10.3390/ijms25084404

    Figure Lengend Snippet: Figure 7. γ-carboxylation of Gas6 induces Mertk degradation independent of phosphorylation. (A) Mutants of the tagged construct were created. K619M, a substitution in the ATP-binding site (underlined) of the Mertk kinase domain, inhibits autophosphorylation by preventing ATP binding and the exchange of phosphate molecules. * Amino acid position for ADAM17 cleavage. (B) Constructs are treated with 10 nM ofGas6-VK for 30 min after a 6 h serum starvation. With the ability to bind ATP and phosphorylate, the WT construct becomes phosphorylated while the kinase dead K619M mutant does not. (C) Mutant constructs were treated with 3 µM of GW280264X (an ADAM17 inhibitor; GW), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a protea- somal inhibitor) for 4 h. Results show that the absence of the K619M C-terminal fragment from the MG132 treatment with the addition of GW indicates the fragment is a product of cleavage. (D) Confocal images indicated that the K619M mutants have more cytoplasmatic c-Mertk than WT Mertk with MG132 treatment. (E) Comparison of the contrasting pathways between Notch Recep- tor and MerTK Signaling Models. In the absence of a ligand, Notch is not cleaved, while MerTK undergoes homeostatic cleavage, leading to proteasomal degradation. Upon ligand binding, Notch is cleaved at the ADAM 17 site, revealing the gamma-secretase site. Subsequent gamma-secretase cleavage releases the Notch intracellular domain, translocating it to the nucleus for transcriptional activation. Conversely, MerTK, upon ligand (Gas6) interaction, is internalized into endosomal compartments and localizes within lysosomes.

    Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

    Techniques: Phospho-proteomics, Construct, Binding Assay, Mutagenesis, Comparison, Ligand Binding Assay, Activation Assay

    Calibration curve of Gas6 (AF885). Comparison of Proseek (A, B) and DuoSet ELISA Dy885 (C). Detection of each concentration of human recombinant Gas6 from 1 pg/ml to 10 000 pg/ml (n = 3). Bars represent standard deviations of the mean of 3 (A) independent measurements (duplicates) (B). Detection of Gas6 by ELISA Dy885 (C).

    Journal: Biotechnology Reports

    Article Title: Proseek single-plex protein assay kit system to detect sAxl and Gas6 in serological material of brain tumor patients

    doi: 10.1016/j.btre.2018.e00252

    Figure Lengend Snippet: Calibration curve of Gas6 (AF885). Comparison of Proseek (A, B) and DuoSet ELISA Dy885 (C). Detection of each concentration of human recombinant Gas6 from 1 pg/ml to 10 000 pg/ml (n = 3). Bars represent standard deviations of the mean of 3 (A) independent measurements (duplicates) (B). Detection of Gas6 by ELISA Dy885 (C).

    Article Snippet: A linear serial dilution of a human Gas6 standard AF885 from R&D systems was generated between 1 pg/ml and 10 ng/ml and used for the amplification.

    Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Concentration Assay, Recombinant

    Concentrations of Axl and Gas6 in human serum samples during treatment by using Proseek. The calculations were performed according to the calibration curve in for Gas6 and for Axl using the formula y = kx + d. X-axis: P02, P04, P05 serum samples from human patients till recurrence (S). Red dot: serum of a healthy person. y-axis: protein concentration in pg/ml.

    Journal: Biotechnology Reports

    Article Title: Proseek single-plex protein assay kit system to detect sAxl and Gas6 in serological material of brain tumor patients

    doi: 10.1016/j.btre.2018.e00252

    Figure Lengend Snippet: Concentrations of Axl and Gas6 in human serum samples during treatment by using Proseek. The calculations were performed according to the calibration curve in for Gas6 and for Axl using the formula y = kx + d. X-axis: P02, P04, P05 serum samples from human patients till recurrence (S). Red dot: serum of a healthy person. y-axis: protein concentration in pg/ml.

    Article Snippet: A linear serial dilution of a human Gas6 standard AF885 from R&D systems was generated between 1 pg/ml and 10 ng/ml and used for the amplification.

    Techniques: Protein Concentration

    Characteristics of patients at inclusion

    Journal: Critical Care

    Article Title: Growth arrest-specific protein 6 plasma concentrations during septic shock

    doi: 10.1186/cc5158

    Figure Lengend Snippet: Characteristics of patients at inclusion

    Article Snippet: Three additional washes were then performed and 100 μl plasma or standards (recombinant human Gas6; RnD Systems) were added for two hours at room temperature.

    Techniques:

    Correlation between plasma Gas6 concentration and SOFA score. (a) SOFA scores were separated into quartiles and plotted against Gas6 plasma concentration. Groups were compared using the Mann-Whitney U test. (b) Individual SOFA scores were plotted against Gas6 concentration and the correlation was evaluated using the Spearman test (rs = 0.37, P = 0.01). Gas6, growth arrest-specific gene 6 product; SOFA, Sepsis-related Organ Failure Assessment.

    Journal: Critical Care

    Article Title: Growth arrest-specific protein 6 plasma concentrations during septic shock

    doi: 10.1186/cc5158

    Figure Lengend Snippet: Correlation between plasma Gas6 concentration and SOFA score. (a) SOFA scores were separated into quartiles and plotted against Gas6 plasma concentration. Groups were compared using the Mann-Whitney U test. (b) Individual SOFA scores were plotted against Gas6 concentration and the correlation was evaluated using the Spearman test (rs = 0.37, P = 0.01). Gas6, growth arrest-specific gene 6 product; SOFA, Sepsis-related Organ Failure Assessment.

    Article Snippet: Three additional washes were then performed and 100 μl plasma or standards (recombinant human Gas6; RnD Systems) were added for two hours at room temperature.

    Techniques: Concentration Assay, MANN-WHITNEY

    Plasma Gas6 concentrations according to need for CVVH. The two groups were compared using the Mann-Whitney U test. CVVH, continuous venovenous haemofiltration; Gas6, growth arrest-specific gene 6 product.

    Journal: Critical Care

    Article Title: Growth arrest-specific protein 6 plasma concentrations during septic shock

    doi: 10.1186/cc5158

    Figure Lengend Snippet: Plasma Gas6 concentrations according to need for CVVH. The two groups were compared using the Mann-Whitney U test. CVVH, continuous venovenous haemofiltration; Gas6, growth arrest-specific gene 6 product.

    Article Snippet: Three additional washes were then performed and 100 μl plasma or standards (recombinant human Gas6; RnD Systems) were added for two hours at room temperature.

    Techniques: MANN-WHITNEY

    Time course of plasma Gas6 in surviving versus nonsurviving patients. Values are expressed as median with interquartile range. Gas6, growth arrest-specific gene 6 product.

    Journal: Critical Care

    Article Title: Growth arrest-specific protein 6 plasma concentrations during septic shock

    doi: 10.1186/cc5158

    Figure Lengend Snippet: Time course of plasma Gas6 in surviving versus nonsurviving patients. Values are expressed as median with interquartile range. Gas6, growth arrest-specific gene 6 product.

    Article Snippet: Three additional washes were then performed and 100 μl plasma or standards (recombinant human Gas6; RnD Systems) were added for two hours at room temperature.

    Techniques: