Antithrombin, rapamycin, A6730, Evans blue, and triphenyltetrazolium chloride (TTC) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-beclin-1 (1:1000), anti-GAPDH (1:5000), anti-protein kinase B (Akt; 1:1000), anti-phosphorylated (p)-Akt (Ser473,1:1000), anti-autophagy protein microtubule-associated protein 1 light chain-3B (LC3B; 1:1000), anti-B-cell lymphoma 2 (Bcl-2; 1:1000), anti-Bcl-2 associated x (Bax; 1:1000), and anti-p62 (1:1000) antibodies were obtained from either Cell Signaling Technology (Danvers, MA) or Abcam (Cambridge, UK). Phenylmethane sulphonyl fluoride (PMSF), the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit, and the bicinchoninic acid protein quantification kit were obtained from Beyotime (Shanghai, China). The creatine kinase (CK) assay kit was purchased from the Jiancheng Bioengineering Institute (Nanjing, China). Goat anti-rabbit secondary antibodies were purchased from Biosharp (Guangzhou, China).
Antithrombin
Manufactured by Merck Group
Sourced in Spain
Antithrombin is a laboratory equipment product manufactured by Merck Group. It is a protein that functions as a natural anticoagulant, playing a crucial role in regulating blood clotting processes.
Automatically generated - may contain errors
Lab products found in correlation
Evans blue, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Anti beclin 1, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid protein quantification kit, by Beyotime (1 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Anti protein kinase b akt, by Cell Signaling Technology (1 mentions)
Anti b cell lymphoma 2 bcl 2, by Cell Signaling Technology (1 mentions)
Phenylmethanesulphonyl fluoride pmsf, by Beyotime (1 mentions)
Terminal deoxynucleotidyl transferase dutp nick end labeling tunel assay kit, by Beyotime (1 mentions)
A6730, by Merck Group (1 mentions)
Enteropeptidase, by Merck Group (1 mentions)
Spectramax paradigm, by Molecular Devices (1 mentions)
96 well plate, by Corning (1 mentions)
Tween 80, by Merck Group (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Cacl2, by Fujifilm (1 mentions)
Pai 1, by ProSpec (1 mentions)
3 protocols using antithrombin
1
Autophagy Regulation in Cardiac Injury
2
Antithrombin-Enteropeptidase Covalent Complexes
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Formation of covalent complexes between antithrombin and enteropeptidase (Sigma, Madrid, Spain) was evaluated by adding an excess of enteropeptidase (150 μM) to 5 μM of antithrombin and incubating the complex at different times (0, 15, 30, 45 and 60 min) at 37 °C. Complexes were evaluated by 8% SDS-PAGE under reducing and non-reducing conditions, as described elsewhere43 (link).
To evaluate which functional domains of antithrombin are required for the inhibition of enteropeptidase, two different antithrombin mutants were used, and enteropeptidase activity was measured (antithrombin London and antithrombin Toyama). Mutagenesis of plasmid containing cDNA coding for antithrombin, transfection of HEK-EBNA cells and purification of variants were carried out as previously described13 (link)15 (link).
To evaluate which functional domains of antithrombin are required for the inhibition of enteropeptidase, two different antithrombin mutants were used, and enteropeptidase activity was measured (antithrombin London and antithrombin Toyama). Mutagenesis of plasmid containing cDNA coding for antithrombin, transfection of HEK-EBNA cells and purification of variants were carried out as previously described13 (link)15 (link).
3
Turbidity Assay and Fibrin Gel Lysis
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The turbidity assay was performed according to a minor modification of the method of Kim et al. 31 at 37°C in a 96-well plate (Corning) on a SpectraMax Paradigm plate reader (Molecular Devices, Sunnyvale). Measurements were performed in quadruplicate. Turbidity was monitored once a minute at a 350-nm wavelength and calculated as the mean value (n = 4) in a volume of 100 μL HBS (HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-buffered saline solution) at pH 7.4.
For fibrin gel formation, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl 2 (Wako Pure Chemical), 0.01% Tween 80 (Sigma-Aldrich), and 0.5 NIH unit/mL thrombin (Sigma-Aldrich) were added to 10 μg/mL 1101 mAb or 3 U/mL anti-thrombin (SLS Behring K) as AT III (antithrombin III).
For the lysis of the fibrin gel, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl 2 (Wako), 0.01% Tween 80 (Sigma-Aldrich), 0.5 NIH unit/mL thrombin (Sigma-Aldrich), 0.2 μM PLG (Enzyme Research Laboratories), and 0.3 nM tPA (Alteplase, Kyowa Hakko Kirin) were added to 10 μg/mL1101 mAb or a mixture of 0.10 μM α2-PI (Hematologic Technologies) and 2.0 ng/mL PAI-1 (ProSpec), and used as a negative control.
For fibrin gel formation, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl 2 (Wako Pure Chemical), 0.01% Tween 80 (Sigma-Aldrich), and 0.5 NIH unit/mL thrombin (Sigma-Aldrich) were added to 10 μg/mL 1101 mAb or 3 U/mL anti-thrombin (SLS Behring K) as AT III (antithrombin III).
For the lysis of the fibrin gel, 2.0 mg/mL fibrinogen (Sigma-Aldrich), 5 mM CaCl 2 (Wako), 0.01% Tween 80 (Sigma-Aldrich), 0.5 NIH unit/mL thrombin (Sigma-Aldrich), 0.2 μM PLG (Enzyme Research Laboratories), and 0.3 nM tPA (Alteplase, Kyowa Hakko Kirin) were added to 10 μg/mL1101 mAb or a mixture of 0.10 μM α2-PI (Hematologic Technologies) and 2.0 ng/mL PAI-1 (ProSpec), and used as a negative control.
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