Journal: Mediators of inflammation

Article Title: Growth Arrest-Specific 6 Enhances the Suppressive Function of CD4 + CD25 + Regulatory T Cells Mainly through Axl Receptor.

doi: 10.1155/2017/6848430

Figure Lengend Snippet: Figure 6: Gas6 modulates the Foxp3 and CTLA4 expression mainly through Axl receptor. (a–d) CD4+CD25+Tregs were treated with anti- Axl or anti-Mertk Abs or PBS in the presence of 100 ng/ml rmGas6. After 24 h of incubation, the expression of CTLA-4 and Foxp3 was determined by flow cytometry (𝑛= 4/group). ∗𝑃< 0.05 compared with the value for the Gas6 group, and #𝑃< 0.05 compared with the value for rmGas6+anti-Mertk group. (e–h) The expression of CTLA-4 and Foxp3 in Tregs with or without Axl knockout was determined by flow cytometry. ∗𝑃< 0.05 compared with the value for the Gas6 group.

Article Snippet: To investigate the effect of Gas6 on CD4+CD25+Tregs in vivo, healthy mice were administered 1, 3, or 6 μg/mouse of rmGas6 (8310-GS, R&D Systems, Minneapolis, MN) via tail vein.

Techniques: Expressing, Incubation, Flow Cytometry, Knock-Out

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

doi: 10.1159/000373940

Figure Lengend Snippet: Fig. 2. Gas6 delays the senescence process in VSMCs. (A and B) Western blots demonstrating that cells trea- ȋʹͷͲȀȌ͵ϐ ͳ Ͷ and p21Cip1 expression. (C) The ef- fects of Gas6 on the IS and RS models were examined using western blotting. In the IS model, the Gas6-tre- ated cells showed low p21Cip1 and p16 Ͷ expression. In the RS model, the Gas6-treated cells also showed low p21Cip1 and p16 ͶǤȋȌǦȾǦ Ǧ ϐ Ǧ Ǧ RS models. (E and F) When these cells were treated with Axl-Fc, the levels of p16 Ͷ and p21Cip1 and the ǦȾǦ ϐ Ǥ (n=3 in each case). The values are presented as the mean±SD. *P<0.05, **P<0.01 and ***P<0.001 compared with the corresponding blank group; #P<0.05, ##P<0.01 and ###P<0.001 compared with the corresponding blank group. Bar, 200 μm.

Article Snippet: Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA).

Techniques: Western Blot, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

doi: 10.1159/000373940

Figure Lengend Snippet: Fig. 3. Axl is the primary receptor in the Gas6-mediated anti-senescence effect. (A) Western blotting and SA- ȾǦͳ Ͷ and p21Cip1ϐ in R428-treated cells regardless of Gas6 treatment compared with the two R428-free cell groups, whereas ϐ ͶʹͺǦ ǤȋȌǦȾǦ ͶʹͺǦ ϐ compared with the two R428-free cell groups. All the results shown are from representative experiments (n=3 in each case). The values are presented as the mean±SD. **P<0.01 and ***P<0.001 compared with the non-R428-treated group; ##P<0.01 and ###P<0.001 compared with the non-R428-treated group. Bar, 200 μm.

Article Snippet: Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA).

Techniques: Western Blot

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

doi: 10.1159/000373940

Figure Lengend Snippet: Fig. 4. Gas6 promotes the transition from G1 to S phase. Cell cycle analyses showing that Gas6-treated cells in both the IS and the RS models showed higher percentages of S phase and a lower percentage of G1 phase Ǧ Ǧ Ǣȋ Ȍϐ with the corresponding controls. (B) EdU staining results showed that the positive staining rates in both the IS and the RS models after Gas6 treatment were higher than in controls. All the results shown are from representative experiments (n=3 in each case). The values are presented as the mean±SD. *P<0.05 compared with the non-Gas6-treated group; #P<0.05 compared with the non-Gas6-treated group.

Article Snippet: Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA).

Techniques: Staining

Journal: Virology

Article Title: Inhibition of type I interferon responses by adenovirus serotype-dependent Gas6 binding

doi: 10.1016/j.virol.2017.12.016

Figure Lengend Snippet: (A.) Detection of Gas6 in virion density fractions (HAdV-5C = 1.21:1.43 g/mL; VSV = 1.11–1.21 g/mL) isolated from 20/40/80% histodenz step gradient purified samples by Immunoblotting. (B.) Detection of Gas6 bound to HAdV-5C capsid proteins, HAdV-5C particles or individual capsid proteins were separated by SDS-PAGE under non-reducing or denaturing conditions prior to transfer to nitrocellulose membranes. Membranes were overlaid with Gas6 medium and, bound Gas6 was detected by Immunoblotting. Total protein from each lane corresponds to gels ran in duplicate and stained with Coomassie Blue. (C.) Micro titer wells coated with (1 × 10−5−1 μg) of HAdV-5C or HAdV-5CF16 were overlaid with Gas6 medium. The concentration of AdV particles per well or bound Gas6 was measured by ELISA with antibodies specific to HAdV-5C hexon protein (9c12) or Gas6.

Article Snippet: Reagents hGas6 conditioned media was isolated from Hela-VKORC1-hGas6 cells grown in serum-free media with or without 2uM warfarin for 72 h. Purified recombinant human Gas6 and recombinant mouse Gas6 were purchased from R&D Systems (885-GSB-050 and 8310-GS-050).

Techniques: Isolation, Purification, Western Blot, SDS Page, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Virology

Article Title: Inhibition of type I interferon responses by adenovirus serotype-dependent Gas6 binding

doi: 10.1016/j.virol.2017.12.016

Figure Lengend Snippet: (A.) 1 μg of AdV (HAdV-5C, HAdV-2C, HAdV-12A, HAdV-5CF16 or HAdV-28D) particles or no treatment control were immobilized on Polyvinyl pyrrolidon (PVDF) membranes and exposed to Gas6 medium using a dot blot apparatus, and followed by Immunoblotting with Abs against hGas6. Blot intensities were measured using ImageJ software with densitometry indicated as optical density (O.D.). (B.) Phylogenetic tree based on alignment of fiber protein amino acid sequence. Cellular receptor use based on Ad class is noted. (C.) 1 μg of purified recombinant Ad5 or Ad28 fiber knob proteins or authentic HAdV-5C virions were separated by SDS-PAGE under non-reducing or denaturing conditions prior to transfer to nitrocellulose membranes. Membranes were overlaid with Gas6 medium and, bound Gas6 was detected by Immunoblotting. Total protein from fiber knob lanes corresponds to gels ran in duplicate and stained with Coomassie Blue.

Article Snippet: Reagents hGas6 conditioned media was isolated from Hela-VKORC1-hGas6 cells grown in serum-free media with or without 2uM warfarin for 72 h. Purified recombinant human Gas6 and recombinant mouse Gas6 were purchased from R&D Systems (885-GSB-050 and 8310-GS-050).

Techniques: Control, Dot Blot, Western Blot, Software, Sequencing, Purification, Recombinant, SDS Page, Staining

Journal: Virology

Article Title: Inhibition of type I interferon responses by adenovirus serotype-dependent Gas6 binding

doi: 10.1016/j.virol.2017.12.016

Figure Lengend Snippet: (A.) Hela cells constructed to stably express vitamin K 2,3-epoxide (VKORC1) and human Gas6 (hGas6) were grown in serum-free media with or without 2 μM warfarin for 72 h. The Conditioned media were collected and resolved by SDS-PAGE along with commercially available purified hGas6 (R&D systems) followed by Immunoblotting with Abs against hGas6 and γ-carboxyglutamic acid (Sekisui Diagnostics). Sample preparations were performed under reducing conditions. (B.) 1 μg HAdV-5C was resolved by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were overlaid with different Gas6 mediums, described above, and followed by Immunoblotting with Abs against hGas6. Band intensities were measured using ImageJ software with densitometry indicated as optical density (O.D.).

Article Snippet: Reagents hGas6 conditioned media was isolated from Hela-VKORC1-hGas6 cells grown in serum-free media with or without 2uM warfarin for 72 h. Purified recombinant human Gas6 and recombinant mouse Gas6 were purchased from R&D Systems (885-GSB-050 and 8310-GS-050).

Techniques: Construct, Stable Transfection, SDS Page, Purification, Western Blot, Membrane, Software

Journal: Virology

Article Title: Inhibition of type I interferon responses by adenovirus serotype-dependent Gas6 binding

doi: 10.1016/j.virol.2017.12.016

Figure Lengend Snippet: (A.) 1 μg of AdV (HAdV-5C or HAdV-28D) particles or no treatment control were immobilized on Polyvinyl pyrrolidon (PVDF) membranes and exposed to Gas6 medium using a dot blot apparatus, and followed by Immunoblotting with Abs against. Blot intensities were measured using ImageJ software with densitometry indicated as optical density (O.D.) (B-C.) J774-dual reporter cells were stimulated with HAdV-5C (3000vp/cell), HAdV-28D (3000vp/cell), or Poly I:C (10 μg/mL) in the presence or absence (−) of Gas6 (0, 20, 200 or 2000 ng/mL) for 20 h. Cell supernatants were collected and luciferase activity was measured using a luminometer and expresses as relative light units (RLU). (C.) Relative IFN response was calculated by normalizing RLU values from 200 ng/mL Gas6 treatments to no Gas6 control treatments. Results depict the average and standard error of the mean across three independent experiments performed in triplicate. (D.) Murine Bone Marrow Derived Macrophage (BMDM) cells were stimulated with HAdV-5C (3000vp/cell), HAdV-28D (3000vp/cell), or Poly I:C (10 μg/mL) in the presence or absence (−) of Gas6 (200 ng/mL) for 20 h. Cell supernatants were collected and IFNβ levels were measured by ELISA. Relative IFN response was calculated by normalizing pg/mL IFN beta to no Gas6 control treatments. Error bars depict standard error of the mean across three samples. Significant differences between control and Gas6 stimulation are denoted (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). (E.) J774-dual cells were transduced with HAdV-5C expressing a GFP transgene (AdV5-GFP) at 3000vp/cell in the presence or absence of 200 ng/mL Gas6. GFP expression was measured by flow cytometry after 20 h and the mean fluorescent intensities (MFI) plotted.

Article Snippet: Reagents hGas6 conditioned media was isolated from Hela-VKORC1-hGas6 cells grown in serum-free media with or without 2uM warfarin for 72 h. Purified recombinant human Gas6 and recombinant mouse Gas6 were purchased from R&D Systems (885-GSB-050 and 8310-GS-050).

Techniques: Control, Dot Blot, Western Blot, Software, Luciferase, Activity Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transduction, Expressing, Flow Cytometry

Journal: Virology

Article Title: Inhibition of type I interferon responses by adenovirus serotype-dependent Gas6 binding

doi: 10.1016/j.virol.2017.12.016

Figure Lengend Snippet: (A-B.) THP-1 cells were transduced with HAdV-5C (3000vp/cell) encoding a luciferase transgene (AdV5-luc) in the presence or absence of Gas6 (200 ng/mL). Cell lysates were collected at indicated time points (0–96 h) following transduction and luciferase expression was measured using a luminometer and expressed as relative light units (RLU) (B.) Average fold change in RLUs compared to no Gas6 control across three independent experiments each performed in triplicate. Error bars depict standard error of the mean of the averages of three independent experiments. Significant differences between control and Gas6 stimulation are denoted (* = p < 0.05, ** = p < 0.01).

Article Snippet: Reagents hGas6 conditioned media was isolated from Hela-VKORC1-hGas6 cells grown in serum-free media with or without 2uM warfarin for 72 h. Purified recombinant human Gas6 and recombinant mouse Gas6 were purchased from R&D Systems (885-GSB-050 and 8310-GS-050).

Techniques: Transduction, Luciferase, Expressing, Control