Journal: Journal of Inflammation (London, England)

Article Title: The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization

doi: 10.1186/s12950-015-0081-4

Figure Lengend Snippet: GW9662 induces M2c-like cells that upregulate MerTK and its ligand Gas6. (A-C) Healthy monocytes were cultured in serum-free medium in the absence of cytokines or growth factors (M0 differentiation), with or without the PPAR-γ antagonist GW9662 (2.5-10 μM), for 4 days; when specified, the PPAR-γ agonist rosiglitazone (1 μM) was added. Expression of MerTK, CD163 and CD16 was measured by flow cytometry. (D-E) Gas6 production levels were quantified by ELISA in culture medium, upon incubation with or without GW9662 (2.5-10 μM) of otherwise untreated cells (M0 conditions), LPS (50 ng/ml; M1 conditions) or IL-4 (20 ng/ml; M2a conditions) exposed cells. (A-E) Pooled data are represented as mean values ± SEM. Analysis was performed using one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. When not specified by additional graphic signs, statistical annotations (asterisks) refer to comparisons with respect to the relative GW9662 untreated control group. Each set of data is representative of three independent experiments.

Article Snippet: Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection.

Techniques: Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Growth arrest-specific protein 6 (Gas6) pretreatment inhibits transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in lung epithelial cells (ECs). ( A – C ) LA-4 and ATII ECs were pretreated with 400 ng/mL Gas6 for 20 h prior to 10 ng/mL TGF-β1 treatment for 48 or 72 h. ( A ) Morphological changes in LA-4 ECs were examined by phase-contrast microscopy. Scale bars = 50 μm. Results are representative of three independent experiments. ( B ) Immunoblots of total cell lysates were performed with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies. Densitometry of the relative abundances of the indicated EMT markers. Alpha-tubulin was used as a control. ( C ) The amount of EMT markers’ mRNAs in cell lysates was analysed by real-time PCR and normalized to that of hypoxanthine phosphoribosyltransferase. Values represent the mean ± S.E. of three independent experiments. * P < 0.05; compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Microscopy, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Growth arrest-specific protein 6 (Gas6) pretreatment reduces epithelial-mesenchymal transition (EMT)-regulating transcription factor expression and blocks Smad-independent transforming growth factor (TGF)-β1 signalling in epithelial cells. ( A – C ) LA-4 and ATII epithelial cells (ECs) were pretreated with 400 ng/mL Gas6 20 h prior to 10 ng/mL TGF-β1 stimulation for 48 or 72 h. ( A , B ) The amounts of Snai1/2, Zeb1/2, and Twist1 mRNA were analysed by real-time PCR and normalized to that of hypoxanthine phosphoribosyltransferase ( Hprt ). ( C , D ) Representative immunoblots of LA-4 EC lysates were performed with anti-Snail1, -Zeb1, -total/phosphorylated ERK1/2, and -Akt protein antibodies. Beta-actin or alpha-tubulin was used as a loading control. Densitometric analysis of the indicated protein abundances. Data in all bar graphs are the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Cyclooxygenase (COX)-2 signaling is required for growth arrest-specific protein 6 (Gas6)-induced production of prostaglandin (PG)E 2 , PGD 2 , and their receptors. ( A – C ) LA-4 and primary alveolar type II (AT II) epithelial cells (ECs) were treated with 400 ng/mL Gas6 for the times indicated. ( A ) qPCR analysis of Cox2 and Cox1 mRNAs in cell lysates. ( B ) Representative immunoblots of LA-4 EC lysates were performed with anti-COX-2, -COX-1, or -α-tubulin antibodies. ( C ) PGE 2 or PGD 2 levels in conditioned media from LA-4 and AT II ECs were measured by enzyme immunoassay. ( D ) Immunoblots of total cell lysates were performed with anti-COX-2 antibodies in LA-4 ECs transfected with COX-2 specific or control siRNA for 6 h. Densitometric analysis of the COX-2 relative abundances. PGE 2 and PGD 2 levels in conditioned media from LA-4 ECs transfected with COX-2 specific or control siRNA for 6 h prior to treatment with 400 ng/mL Gas6 for 20 h were measured by EIA. ( E ) qPCR analysis of Ptger2, Ptger4, Dp1, and Dp2 mRNA in LA-4 ECs treated with 400 ng/mL Gas6 for the time indicated. ( F ) Immunoblot analysis of EP2, EP4, DP1, or DP2 in LA-4 cells. Densitometric analysis of the indicated receptor’ relative abundances. ( G ) qPCR analysis of Ptger2, Ptger4, Dp1, and Dp2 mRNA in LA-4 ECs transfected with COX-2 specific or control siRNA for 6 h prior to treatment with 400 ng/mL Gas6 for 20 h. ( H ) qPCR analysis of Ptger2, Ptger4, Dp1, and Dp2 mRNA in ATII ECs treated with 400 ng/mL Gas6 for the time indicated. Data in all bar graphs are the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Cyclooxygenase (COX)-2-derived signaling mediates growth arrest-specific protein 6 (Gas6)-induced epithelial-mesenchymal transition (EMT) inhibition. ( A – D ) LA-4 ECs were pretreated with 10 μM NS-398 1 h before 400 ng/mL Gas6 treatment for 20 h and then stimulated with 10 ng/mL TGF-β1 treatment for 72 h. ( A ) Morphological changes in the cells were examined by phase-contrast microscopy. Scale bars = 50 μm. ( B ) Immunoblots of total cell lysates were performed with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies. Densitometric analysis of the indicated EMT markers’ relative abundances. ( C , D ) Primary AT II cells were pretreated with 10 μM NS-398 1 h before 400 ng/mL Gas6 treatment for 20 h and then stimulated with 10 ng/mL TGF-β1 treatment for 72 h. qPCR analysis of the mRNAs of EMT markers and EMT-regulating transcription factors. ( E , F ) LA-4 ECs were transfected with COX-2-specific or control siRNAs for 6 h prior to treatment with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 72 h or the times indicated. Representative immunoblots of LA-4 EC lysates were performed with anti-E-cadherin, -N-cadherin, -α-SMA, -total/phosphorylated ERK1/2, and -Akt protein antibodies. Densitometric analysis of the indicated protein abundances. Data in all bar graphs are the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Inhibition, Microscopy, Western Blot, Transfection, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Prostaglandin (PG)E 2 and PGD 2 secretion inhibits growth arrest-specific protein 6 (Gas6)-induced epithelial-mesenchymal transition (EMT) via their receptors. ( A – D ) LA-4 and AT II epithelial cells (ECs) were stimulated with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 48 or 72 h with or without antagonists of EP2 (AH-6809), EP4 (AH-23848), DP1 (BW-A868C), or DP2 (BAY-u3405), each at a dose of 10 μM. ( A ) Representative immunoblots of total cell lysates were performed with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies. ( B ) qPCR analysis of the mRNAs of EMT transcription factors in LA-4 ECs. ( C , D ) qPCR analysis of the mRNAs of EMT markers and EMT transcription factors in primary AT II ECs. Values represent the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Activation of Axl or Mer mediates growth arrest-specific protein 6 (Gas6)-induced inhibition of COX-2 signaling and epithelial-mesenchymal transition (EMT) in LA-4 epithelial cells (ECs). ( A , B ) Immunoblot of total cell lysates were performed with anti-total/phosphorylated Axl and -Mer antibodies in LA-4 ECs treated with 400 ng/mL Gas6 for the times indicated. Densitometric analysis of the indicated protein abundances. ( C , D ) Immunoblots of total cell lysates were performed with anti-Axl, or -Mer antibodies in LA-4 ECs transfected with Axl, Mer, or control siRNA. Densitometric analysis of the indicated protein abundances. ( E – G ) LA-4 ECs were transfected with Axl, Mer, or control siRNAs for 48 h and then stimulated with 400 ng/mL Gas6. ( E , G ) qPCR analysis of the mRNAs of Cox2, Ptger2, Ptger4, Dp1, and Dp2 in LA-4 EC lysates 1 or 20 h after Gas6 stimulation. ( F ) PGE 2 and PGD 2 levels in conditioned media 20 h after Gas6 stimulation were measured by enzyme immunoassay. ( H – J ) LA-4 ECs were transfected with Axl, Mer, or control siRNAs for 48 h prior to treatment with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 72 h or the times indicated. ( H ) Representative immunoblots of total cell lysates with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies in the indicated samples. ( I ) qPCR analysis of the mRNAs of EMT transcription factors. ( J ) Representative immunoblots of total cell lysates with anti-total/phosphorylated ERK1/2 and -Akt protein antibodies. Values represent the mean ± S.E. of three independent experiments. * P < 0.05; compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Inhibition, Western Blot, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Growth arrest-specific protein 6 (Gas6)/Axl signaling inhibits migration and invasion of LA-4 and alveolar type II (AT II) epithelial cells (ECs) via prostaglandin (PG)E 2 and PGD 2 . ( A – D ) LA-4 and primary AT II ECs were stimulated with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 with or without antagonists of EP2 (AH-6809), EP4 (AH-23848), DP1 (BW-A868C), or DP2 (BAY-u3405), each at a dose of 10 μM, for 48 or 72 h. The quantification of migrated or invaded cells in Boyden chambers. ( E , F ) LA-4 ECs were transfected with Axl, Mer, or control siRNAs for 48 h prior to treatment with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 72 h. The cells were visualized by phase-contrast microscopy for the analysis of migratory in ( E ) left and invasive in ( F ) left abilities using Fn-coated Transwell and Matrigel-coated Transwell plates, respectively. Scale bars: 100 μm. Quantification of cells that migrated in ( E ) right or invaded in ( F ) right. Values represent the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Transfection, Control, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Crosstalk between Akt and NF‐κB pathway mediates inhibitory effect of gas6 on monocytes‐endothelial cells interactions stimulated by P. gingivalis ‐LPS

doi: 10.1111/jcmm.15430

Figure Lengend Snippet: Effect of gas6 in HUVECs on chemotaxis and adhesion between monocytes and endothelial cells stimulated by P. gingivalis ‐LPS. (A‐B) Western blotting for checking efficiency of gas6 transfection in HUVECs. (C‐D) expression of chemokines MCP‐1 and IL‐8 in HUVECS transfected with gas6 siRNA or plasmids, followed with 1 μg/mL P. gingivalis ‐LPS infection for 24 hours. Expression level were detected by ELISA method. * P < .05, ** P < .01 and *** P < .001 vs indicated control groups. (E) representative images (3 independent experiments) showing monocytes recruited by endothelial cells. Gas6 siRNA or plasmid were transfected into HUVECs in the lower chamber of transwell inserts. HUVECs were challenged with 1 μg/mL P. gingivalis ‐LPS for 24 hours, images were captured 3 hours after Calcein AM pre‐labelled THP‐1 cells were added into the upper chamber. Scale bars, 200 μm. (F‐I) Western blotting for detection of adhesion molecules ICAM‐1 and E‐selectin in HUVECS transfected with gas6 siRNA or plasmids, followed with 1 μg/mL P. gingivalis ‐LPS infection for 24 hours. * P < .05 vs indicated control groups. (J) representative images (3 independent experiments) showing monocytes adhering to the surfaces of HUVECs. HUVECs were transfected with gas6 siRNA or plasmid, followed with 1 μg/mL P. gingivalis ‐LPS stimulation for 24 hours. THP‐1 cells were co‐cultured with HUVECs for 3 hours, images were captured after non‐adherent monocytes were removed gently with PBS for 3 times. Scale bar, 200 μm. Unpaired Student's t test was performed (C‐D, H‐I)

Article Snippet: The primal antibodies used in this study were as follows: Phospho‐Akt (Ser473) Rabbit mAb, NF‐κB p65(D14E12) Rabbit mAb, GAS6 (D3A3G) Rabbit mAb, Phospho‐NF‐κB p65 (Ser536) Rabbit mAb, CD54/ICAM‐1 Rabbit Antibody, GAPDH (D16H11) Rabbit mAb (CST), Anti‐pan‐AKT Rabbit Antibody (Abcam), Rabbit Anti‐AXL Polyclonal Antibody, Rabbit Anti‐E‐selectin Polyclonal Antibody (Bioss), TYRO3 Polyclonal Antibody Rabbit (Abclonal) and the Rabbit MERTK Antibody (CUSABIO).

Techniques: Chemotaxis Assay, Western Blot, Transfection, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Crosstalk between Akt and NF‐κB pathway mediates inhibitory effect of gas6 on monocytes‐endothelial cells interactions stimulated by P. gingivalis ‐LPS

doi: 10.1111/jcmm.15430

Figure Lengend Snippet: Akt/NF‐κB pathway mediated the gas6 inhibitory effect. (A) Western blotting for detection of Tyro3 receptor in HUVECs, THP‐1 cells were set as positive control. (B)Western blotting for detecting change of ICAM‐1 and E‐selectin protein level in HUVECs pre‐incubated with selective small molecular inhibitors of Axl and Mer receptor, R428 (10 μg/mL) and UNC2025 (10 μM), respectively, followed by challenged with 1 μg/mL P. gingivalis ‐LPS. (C‐D) change of phosphorylated p65 or Akt level when gas6 in HUVECs was knock‐down or overexpressed, followed with 1 μg/mL P. gingivalis ‐LPS stimulation for 3 hours. (E‐F) change of phosphorylated p65 or Akt level in HUVECs pre‐treated with 30 μM LY294002 for 1 hour and stimulated by 1 μg/mL P. gingivalis ‐LPS for 3 hours. (G‐H) expression of phosphorylated Akt in HUVECs challenged with 0 μg/mL, 0.1 μg/mL, 1 μg/mL and 10 μg/mL for 3 hours. (I‐J) change of phosphorylated Akt level in HUVECs pre‐treated with 100 μM PDTC for 1 hour and challenged with 1 μg/mL P. gingivalis ‐LPS. (K‐L) expression of phosphorylated p65 and Akt in HUVECs pre‐treated with 400 ng/mL recombinant human gas6 protein for 1 hour and stimulated with 1 μg/mL P. gingivalis ‐LPS for 3 hours. (M) mRNA level of ICAM‐1, E‐selectin, MCP‐1 and IL‐8 in HUVECs pre‐treated with 400ng/mL recombinant human gas6 protein for 1 hour, followed by stimulation with 1 μg/mL P. gingivalis ‐LPS for 24 hours. One‐way ANOVA analysis was performed. ( * P < .05, ** P < .01 and *** P < .001)

Article Snippet: The primal antibodies used in this study were as follows: Phospho‐Akt (Ser473) Rabbit mAb, NF‐κB p65(D14E12) Rabbit mAb, GAS6 (D3A3G) Rabbit mAb, Phospho‐NF‐κB p65 (Ser536) Rabbit mAb, CD54/ICAM‐1 Rabbit Antibody, GAPDH (D16H11) Rabbit mAb (CST), Anti‐pan‐AKT Rabbit Antibody (Abcam), Rabbit Anti‐AXL Polyclonal Antibody, Rabbit Anti‐E‐selectin Polyclonal Antibody (Bioss), TYRO3 Polyclonal Antibody Rabbit (Abclonal) and the Rabbit MERTK Antibody (CUSABIO).

Techniques: Western Blot, Positive Control, Incubation, Knockdown, Expressing, Recombinant

Journal: Journal of Cellular and Molecular Medicine

Article Title: Crosstalk between Akt and NF‐κB pathway mediates inhibitory effect of gas6 on monocytes‐endothelial cells interactions stimulated by P. gingivalis ‐LPS

doi: 10.1111/jcmm.15430

Figure Lengend Snippet: Effects of P. gingivalis ‐LPS on expression of gas6 and its receptors in HUVECs. (A‐E) mRNA and protein level of gas6, Axl and Mer in HUVECs challenged with 0 μg/mL, 0.1 μg/mL, 1 μg/mL and 10 μg/mL P. gingivalis ‐LPS for 24 hours. (F‐G) Western blot assay for detecting gas6 protein levels in healthy (H1‐H6) and inflamed (I1‐I4) periodontal tissues. Note that H2 and I1 tissue are from the same patient at the same time. Unpaired Student's t test was performed (B‐G). (* P < .05, ** P < .01 and *** P < .001)

Article Snippet: The primal antibodies used in this study were as follows: Phospho‐Akt (Ser473) Rabbit mAb, NF‐κB p65(D14E12) Rabbit mAb, GAS6 (D3A3G) Rabbit mAb, Phospho‐NF‐κB p65 (Ser536) Rabbit mAb, CD54/ICAM‐1 Rabbit Antibody, GAPDH (D16H11) Rabbit mAb (CST), Anti‐pan‐AKT Rabbit Antibody (Abcam), Rabbit Anti‐AXL Polyclonal Antibody, Rabbit Anti‐E‐selectin Polyclonal Antibody (Bioss), TYRO3 Polyclonal Antibody Rabbit (Abclonal) and the Rabbit MERTK Antibody (CUSABIO).

Techniques: Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Crosstalk between Akt and NF‐κB pathway mediates inhibitory effect of gas6 on monocytes‐endothelial cells interactions stimulated by P. gingivalis ‐LPS

doi: 10.1111/jcmm.15430

Figure Lengend Snippet: The inhibitory effect of P. gingivalis ‐LPS on gas6 expression in HUVECs is mediated by LncRNA GAS6‐AS2 down‐regulation. (A‐C) inhibited mRNA and protein expression of gas6 caused by 1μg/mL P. gingivalis ‐LPS was reduced by pre‐treating HUVECs with 100 μM PDTC for 1 hour. (D) expression of gas6 mRNA, GAS6‐AS1 and GAS6‐AS2 in HUVECs after stimulated with 1 μg/mL P. gingivalis ‐LPS for 1, 3, 6, 9 and 12 hours. (E‐F) Expression of GAS6‐AS1 and GAS6‐AS2 in HUVECs after pre‐treatment with PDTC for 1 hour and stimulation with 1 μg/mL P. gingivalis ‐LPS for 24 hours. (G) expression of gas6 in HUVECs after GAS6‐AS2 was knocked down with shRNA. (H) expression of GAS6‐AS2 in HUVECs after gas6 was knocked down or overexpressed. One‐way ANOVA analysis was performed (B‐F). Unpaired Student's t test was performed (G‐H). (ns: not significant, * P < .05, ** P < .01 and *** P < .001)

Article Snippet: The primal antibodies used in this study were as follows: Phospho‐Akt (Ser473) Rabbit mAb, NF‐κB p65(D14E12) Rabbit mAb, GAS6 (D3A3G) Rabbit mAb, Phospho‐NF‐κB p65 (Ser536) Rabbit mAb, CD54/ICAM‐1 Rabbit Antibody, GAPDH (D16H11) Rabbit mAb (CST), Anti‐pan‐AKT Rabbit Antibody (Abcam), Rabbit Anti‐AXL Polyclonal Antibody, Rabbit Anti‐E‐selectin Polyclonal Antibody (Bioss), TYRO3 Polyclonal Antibody Rabbit (Abclonal) and the Rabbit MERTK Antibody (CUSABIO).

Techniques: Expressing, shRNA

Journal: Journal of Cellular and Molecular Medicine

Article Title: Crosstalk between Akt and NF‐κB pathway mediates inhibitory effect of gas6 on monocytes‐endothelial cells interactions stimulated by P. gingivalis ‐LPS

doi: 10.1111/jcmm.15430

Figure Lengend Snippet: Schematic representation for mechanisms of bi‐directional regulation between gas6 and P. gingivalis ‐LPS in HUVECs. Expression of MCP‐1, IL‐8, ICAM‐1 and E‐selectin induced by P. gingivalis ‐LPS was inhibited by gas6 via Akt/NF‐κB pathway; Gas6 expression in HUVECs was inhibited by P. gingivalis ‐LPS through NF‐κB/GAS6‐AS2 pathway

Article Snippet: The primal antibodies used in this study were as follows: Phospho‐Akt (Ser473) Rabbit mAb, NF‐κB p65(D14E12) Rabbit mAb, GAS6 (D3A3G) Rabbit mAb, Phospho‐NF‐κB p65 (Ser536) Rabbit mAb, CD54/ICAM‐1 Rabbit Antibody, GAPDH (D16H11) Rabbit mAb (CST), Anti‐pan‐AKT Rabbit Antibody (Abcam), Rabbit Anti‐AXL Polyclonal Antibody, Rabbit Anti‐E‐selectin Polyclonal Antibody (Bioss), TYRO3 Polyclonal Antibody Rabbit (Abclonal) and the Rabbit MERTK Antibody (CUSABIO).

Techniques: Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Analysis of Gas6 in Human Platelets and Plasma

doi: 10.1161/01.atv.0000163845.07146.48

Figure Lengend Snippet: Figure 1. Gas6 is undetectable in platelets but present in human plasma. (A) Immunoprecipitation of 100 ng rhGas6 (lane 1), 120 L human plasma (lane 2), supernatant from 5108 iso- lated platelets activated by thrombin (lane 3), lysate of 5108 isolated platelets activated by thrombin (lane 4), lysate of 5108 isolated resting platelets (lane 5). Samples were analyzed by SDS-PAGE under nonreducing conditions and Western blotting using an anti-Gas6 antibody. RhGas6 (100 ng) was used without IP as a control (lane 6). (B) Gas6 in Ba-citrate precipitate of human plasma. Gas6 is virtually absent in the wash fraction (lane 1) but detectable in the redissolved concentrated precipi- tate (lane 2). (C) Protein S in platelets. Human platelets were prepared and activated as described in the text. Following SDS- PAGE under reducing conditions, the transferred protein S was visualized using a polyclonal anti-protein S antibody. Human purified protein S (lane 1 and 2; 100 and 10 ng, respectively), proteolytically processed protein S in the supernatant of thrombin-activated platelets (lane 3), absence of protein S in the lysate of activated platelets (lane 4), uncleaved protein S in rest- ing platelets (lane 5), human plasma (0.5 L; lane 6).

Article Snippet: Goat polyclonal peptide antibodies C-20 and N-20 against Gas6 carboxyand amino-terminus, respectively, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Clinical Proteomics, Immunoprecipitation, Isolation, SDS Page, Western Blot, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Analysis of Gas6 in Human Platelets and Plasma

doi: 10.1161/01.atv.0000163845.07146.48

Figure Lengend Snippet: Figure 3. A typical standard curve of the Gas6 ELISA. The sen- sitivity limit was determined to be 0.4 ng/mL. The assay was linear up to 6.25 ng/mL. The inset represents the linearity range of the assay.

Article Snippet: Goat polyclonal peptide antibodies C-20 and N-20 against Gas6 carboxyand amino-terminus, respectively, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Analysis of Gas6 in Human Platelets and Plasma

doi: 10.1161/01.atv.0000163845.07146.48

Figure Lengend Snippet: Figure 2. Mass spectrometry analysis of partially purified plasma Gas6. Peptide ion masses identified with the primary mass screening are underlined. Peptide sequences confirmed in MS/MS mode are framed. Eight peptide ion masses were chosen for sequencing, all of which confirmed the correct sequence for Gas6.

Article Snippet: Goat polyclonal peptide antibodies C-20 and N-20 against Gas6 carboxyand amino-terminus, respectively, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Mass Spectrometry, Clinical Proteomics, Tandem Mass Spectroscopy, Sequencing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Analysis of Gas6 in Human Platelets and Plasma

doi: 10.1161/01.atv.0000163845.07146.48

Figure Lengend Snippet: Figure 4. Frequency distribution of plasma Gas6 concentration in healthy individuals and the effect of warfarin on plasma Gas6 levels. (A) Citrate- anticoagulated plasma samples of 94 apparently healthy individuals (57 females and 37 males) were tested. Normal distribution was verified by the Kolmogorov–Smirnov test. (B) Citrate-anticoagulated plasma samples of patients on warfarin anticoagulation (n96, 46 females and 50 males) were analyzed. Gas6 levels were found to be significantly decreased in patients (18.0 ng/mL in controls versus 15.4 ng/mL in patients). In addition, plasma Gas6 level decreased progressively with increases in INR (16.6 ng/mL and 14.3 ng/mL in patients with INR 2 to 2.5, and above 2.6, respectively).

Article Snippet: Goat polyclonal peptide antibodies C-20 and N-20 against Gas6 carboxyand amino-terminus, respectively, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Clinical Proteomics, Concentration Assay

Journal: British Journal of Cancer

Article Title: Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma

doi: 10.1038/bjc.2017.354

Figure Lengend Snippet: Characterisation of TAM receptor expression in a panel of human LMS cell lines. ( A ) Lysates from IB112, IB118, IB133, IB134, IB136, SK-LMS-1 and HepG2 (positive control) were analysed by western blot. Beta-actin was used as loading control. Longer exposition is shown for HepG2 with AXL antibody. ( B ) Leiomyosarcoma cells were starved then stimulated with GAS6. After normalisation for protein concentration, phosphotyrosine proteins were immunoprecipitated with a PY20 antibody. The SK-LMS-1 lysate was immunoprecipitated also with an antibody isotype control. Western blot analysis was performed with an anti TYRO3 antibody. ( C ) Graph showing AXL (red), MER (blue) and TYRO3 (green) expression levels in LMS and HepG2 cells by FACS analysis. Results are shown as fold increase compared to isotype control. ( D ) Evaluation of GAS6 levels in LMS cells lysates by ELISA.

Article Snippet: The following day a specific human GAS6 (R&D Systems, Abingdon, UK) kit was used following the manufacturer’s instructions.

Techniques: Expressing, Positive Control, Western Blot, Control, Protein Concentration, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: British Journal of Cancer

Article Title: Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma

doi: 10.1038/bjc.2017.354

Figure Lengend Snippet: TYRO3, AXL and GAS6 expression in sarcomas. ( A ) Representative example of immunohistochemistry on LMS tissues with anti-AXL, anti-TYRO3 and anti-GAS6 antibodies. TYRO3 nuclear expression is shown in detail. Original magnification × 20 and × 40. ( B ) Graph showing the percentage of positive samples from each sarcoma hystotype analysed. Cytoplasmic or nuclear staining was plotted separately. ( C – F ) Gene expression analysis of TYRO3, AXL, GAS6 and PROS1 transcripts, respectively. Data are from gene expression and outcome in 251 sarcoma patients’ samples from ATGsarc microarray database. ( G ) Kaplan–Meier curve for progression-free survival of 94 LMS patients with low or mixed and high expression levels of GAS6 and PROS1 genes cluster. P -values in log-rank test are indicated. n =number of patients in each group. Median PFS are expressed in years.

Article Snippet: The following day a specific human GAS6 (R&D Systems, Abingdon, UK) kit was used following the manufacturer’s instructions.

Techniques: Expressing, Immunohistochemistry, Staining, Gene Expression, Microarray

Journal: Nutrients

Article Title: Vitamin K1 Administration Increases the Level of Circulating Carboxylated Osteocalcin in Critically Ill Patients.

doi: 10.3390/nu17020348

Figure Lengend Snippet: Figure 1. Detection of total Gas6 (A) and γ-carboxylated Gas6 (Gla-Gas6; (B)) in blood plasma of ICU patients before and after vitamin K injection. Gla-Gas6 was quantified in samples all as appropriately diluted to contain equal concentrations of total Gas6. Total Gas6 is significantly increased by vitamin K. Statistical significance was determined using paired t-test; * p = 0.0318, ns = not significant, for comparisons indicated by lines (n = 43).

Article Snippet: In the human Gla-Gas6 ELISA, a 96-well microplate with high protein binding capacity (NuncTM Maxisorp, Thermo Fisher Scientific, Loughborough, UK) was first coated with anti-human Gas6 antibody (1 μg/mL, R&D Systems) diluted in a coating buffer (30 mM Na2CO3, 200 mM NaHCO3, pH 9.0), and the plate was incubated overnight at 4 ◦C.

Techniques: Clinical Proteomics, Injection

Journal: Nutrients

Article Title: Vitamin K1 Administration Increases the Level of Circulating Carboxylated Osteocalcin in Critically Ill Patients.

doi: 10.3390/nu17020348

Figure Lengend Snippet: Figure 4. Correlation analyses were performed between total Gas6, undercarboxylated osteocalcin (ucOC), carboxylated osteocalcin (Gla-OC) and PIVKA-II. The coefficient of determination (r) and statistical significance (p-value) were determined by Pearson correlation analysis, except for Gla-OC data, which were not normally distributed and hence Spearman correlation was applied. N = number of XY pairs representing both ‘before’ and ‘after’ samples from patients. Total Gas6 vs. ucOC (n = 86); total Gas6 vs. Gla-OC (n = 94); total Gas6 vs. PIVKA-II (n = 94); PIVKA-II vs. ucOC (n = 86); PIVKA-II vs. Gla-OC (n = 94). *** p < 0.001, ns = not significant.

Article Snippet: In the human Gla-Gas6 ELISA, a 96-well microplate with high protein binding capacity (NuncTM Maxisorp, Thermo Fisher Scientific, Loughborough, UK) was first coated with anti-human Gas6 antibody (1 μg/mL, R&D Systems) diluted in a coating buffer (30 mM Na2CO3, 200 mM NaHCO3, pH 9.0), and the plate was incubated overnight at 4 ◦C.

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