The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells
2.2. Materials
2.3. Transwell Permeability Assay
2.4. VEC Internalization Assay
2.5. VEC Cleavage Assay
2.6. Western Blotting Assay
2.7. Actin and VEC Immunostaining
2.8. Determination of Length of VEC Membrane Stretches
2.9. Determination of the Intracellular Fraction of VEC
2.10. Statistical Analysis
3. Results
3.1. Human PROS1 Increases Endothelial Cell Monolayer Permeability and Activates the p38 MAPK and Rho/ROCK Pathways
3.2. Human PROS1 Regulates Vascular Endothelial Cadherin (VEC) Internalization and Cleavage
3.3. Implication of Both MERTK and TYRO3 Receptors in PROS1-Induced c-Src and VEC Phosphorylation
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target | Name/Catalog# | Supplier | Type of Antibody | Concentration |
---|---|---|---|---|
Primary antibodies | ||||
Actin | β-actin (13E5) HRP conjugate/#5125 | Cell Signaling Technology (Danvers, MA, USA) | Monoclonal Rabbit IgG | WB 1:1000 |
HSP27 | HSP27 (D6W5V)/#95357 | Cell Signaling Technology | Monoclonal Rabbit IgG | WB 1:1000 |
p-HSP27 | Phospho-HSP27 (Ser82)/#2401 | Cell Signaling Technology | Polyclonal Rabbit AB | WB 1:1000 |
MLC | Myosin Light Chain 2 (D18E2)/#8505 | Cell Signaling Technology | Monoclonal Rabbit IgG | WB 1:1000 |
p-MLC | Phospho-Myosin Light Chain 2 (Thr18/Ser19)/#3674 | Cell Signaling Technology | Polyclonal Rabbit AB | WB 1:1000 |
PAK | PAK1 Antibody/#2602 | Cell Signaling Technology | Polyclonal Rabbit AB | WB 1:1000 |
p-PAK | Phospho-PAK1(Ser144)/PAK2 (Ser141)/#2606 | Cell Signaling Technology | Polyclonal Rabbit AB | WB 1:1000 |
c-Src | Src Antibody/#2108 | Cell Signaling Technology | Polyclonal Rabbit AB | WB 1:1000 |
p-c-Src | Phospho-Src Family (Tyr416)/#2101 | Cell Signaling Technology | Polyclonal Rabbit AB | WB 1:1000 |
VEC | Purified Mouse Anti-Human CD144 (55-7H1)/#555661 | BD Biosciences | Monoclonal Mouse IgG | WB 1:1000 IF 1:400 |
Anti-VE-cadherin AB, (clone BV6) /#MABT134 | Millipore | Monoclonal Mouse IgG | WB 1:1000 | |
p-VEC | Phospho-VEC (Ser665) | * | Polyclonal Rabbit AB | WB 1:1000 |
Phospho-VEC (Tyr 685) | ** | Polyclonal Rabbit AB | WB 1:1000 | |
Secondary antibodies | ||||
- | Anti-Mouse IgG—Peroxidase AB /#A4416 | Sigma-Aldrich (Burlington, MA, USA) | Polyclonal Goat IgG | WB 1:4000/1:5000 |
- | Anti-Rabbit IgG—Peroxidase AB /#A0545 | Sigma-Aldrich | Polyclonal Goat IgG | WB 1:4000/1:5000 |
- | Anti-Mouse/Rabbit IgG Antibody (H+L), Biotinylated/#BA-1400 | Vector Laboratories | Polyclonal Horse IgG | IF 1:100 |
Neutralizing and Irrelevant antibodies | ||||
AXL | Human AXL Antibody/#AF154 | R&D Systems (Minneapolis, MN, USA) | Polyclonal Goat IgG | 10 µg/mL |
MERTK | Human MERTK Antibody/#AF891 | R&D Systems | Polyclonal Goat IgG | 10 µg/mL |
PROS1 | Anti-Human Protein S /#A038401-2 | Agilent Dako (Santa Clara, CA, USA) | Polyclonal Rabbit AB | 10 µg/mL |
TYRO3 | Human Dtk Antibody/#AF859 | R&D Systems | Polyclonal Goat IgG | 10 µg/mL |
- | Normal Rabbit IgG Control/#AB105C | R&D Systems | Polyclonal Rabbit IgG | 10 µg/mL |
- | Normal Goat IgG Control/#AB108C | R&D Systems | Polyclonal Goat IgG | 10 µg/mL |
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Joussaume, A.; Kanthou, C.; Pardo, O.E.; Karayan-Tapon, L.; Benzakour, O.; Dkhissi, F. The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability. Curr. Issues Mol. Biol. 2024, 46, 3278-3293. https://doi.org/10.3390/cimb46040205
Joussaume A, Kanthou C, Pardo OE, Karayan-Tapon L, Benzakour O, Dkhissi F. The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability. Current Issues in Molecular Biology. 2024; 46(4):3278-3293. https://doi.org/10.3390/cimb46040205
Chicago/Turabian StyleJoussaume, Aurélie, Chryso Kanthou, Olivier E. Pardo, Lucie Karayan-Tapon, Omar Benzakour, and Fatima Dkhissi. 2024. "The Vitamin K-Dependent Anticoagulant Factor, Protein S, Regulates Vascular Permeability" Current Issues in Molecular Biology 46, no. 4: 3278-3293. https://doi.org/10.3390/cimb46040205