Journal: Scientific Reports

Article Title: Copper(II)-binding equilibria in human blood

doi: 10.1038/s41598-020-62560-4

Figure Lengend Snippet: Separation of human serum proteins by SEC. 4 x diluted human serum (a), 10 μM Cu•CP ( b ), 10 μM HSA plus 10 μM Cu(II) ( c ), 2.5 μM α2M tetramer plus 10 μM Cu(II) ( d ), 10 μM HSA, 2.5 μM α2M tetramer plus 10 μM Cu(II) ( e ). Conditions: incubation buffer 50 mM HEPES, 50 mM NaCl, pH 7.4; LC-ICP MS: column - 10 × 300 mm Superdex 200; elution buffer 200 mM NH 4 NO 3 , pH 7.4; flow rate 0.4 ml/min; injection volume 10 μl; UV at 280 nm (left axis and black lines) and Cu-63 (right axis and red line), monitored by ICP MS. Figure was created by program Origin 9 Pro ( https://www.originlab.com/ ).

Article Snippet: The experiments were carried out with two preparations of normal (“slow form”) human plasma α2M (from Sigma and Athens Research) as well as with the so-called “fast form” of α2M from Athens Research.

Techniques: Incubation, Injection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of uPA with human plasma α2-macroglobulin (α2M), but not with human recombinant suPAR, results in protection from inactivation by PAI-1. scuPA (50 nM) was incubated with 150 nM suPAR, and 50 nM scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) were incubated with 2.0 μM α2M from human plasma in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C for 7 h. At the end of incubation, complexes of uPA with suPAR or α2M were isolated from each reaction mixture by IP with 20 μg of MAb ATN-658 (ATN-658 does not affect amidolytic activity of uPA complexed with suPAR) and with 20 μg of polyclonal sheep anti-human α2M antibodies. Reaction mixtures were incubated with 40 μl of 1:1 slurry of protein A/G Plus Agarose for 2 h at room temperature on a rocker. Reaction mixtures were centrifuged at 3,000–6,000 rpm using an AccuSpin Micro R centrifuge. The resin was washed 3 times with 0.6 ml of cold HEPES buffer, and 50 μl of the resin suspension (total vol 200 μl) was transferred to 96-well plates. Amidolytic uPA activity in precipitated samples was measured with (white bars) or without (gray bars) 80 nM PAI-1. The composition of each reaction mixture, antibodies used for IP, and presence of PAI-1 are shown in the table under the plot.

Article Snippet: . scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Incubation, Clinical Proteomics, Recombinant, Isolation, Activity Assay, Suspension

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of scuPA with rabbit PFs forms αM complexes possessing uPA amidolytic activity that resists PAI-1. A: IP of uPA activity by anti-rabbit α2M IgY from PFs of rabbits treated with intrapleural ED of scuPA after preincubation with 50 nM scuPA for 5 h at 37°C in 50 mM HEPES/NaOH, pH 7.4. Samples were immunoprecipitated with α2M IgY or control nonspecific IgY, and uPA amidolytic activity was measured with (white) or without (gray) added exogenous PAI-1 (80 nM) as described in materials and methods and in Fig. 5. uPA amidolytic activity was significantly (*P < 0.05) increased in both groups of PFs from rabbits IP with anti-rabbit α2M vs. those IP nonspecific IgY. B: bar plot of the uPA activity in the precipitates from PFs of animals treated with the ED of scuPA or HD of Activase (Act), Abbokinase (Abb), or vehicle (n = 2 randomly selected samples/each paired group). The precipitation was carried out with 40 μg of either anti-rabbit α2M (filled bars) or nonspecific IgY (hatched bars). The complexes were captured with 80 μl of Agarose slurry and analyzed as described in Fig. 5. C: anti-α2M but not anti-uPAR antibodies precipitate uPA activity from PFs of rabbits, treated with ED of scuPA. IP of uPA activity by polyclonal sheep anti-human α2M antibodies and anti-human uPAR MAb (cross-reacting with rabbit αM and rabbit uPAR, respectively) from PFs of rabbits treated with ED of scuPA. PFs were incubated with 40 μg of antibodies for 2 h in 50 mM HEPES/NaOH, pH 7.4, either with anti-uPAR MAb or with anti-α2M antibodies. In control experiments, PFs were supplemented with exogenous mixtures of 150 nM suPAR with either 50 nM HMW or LMW tcuPA before anti-uPAR MAb was added. Another set of controls contained PFs without addition of antibodies. The composition of each reaction mixture and antibodies used for IP are shown in the table under the plot. Complexes were precipitated with 40 μl of Protein A/G Plus Agarose, and uPA amidolytic activity was measured with (white) or without (gray) 80 nM PAI-1 added, as described in Fig. 5 legend. uPA amidolytic activity was significantly (P < 0.05) increased in PFs incubated with sheep anti-human α2M antibodies and samples supplemented with HMW tcuPA complexed with suPAR (activity without PAI-1) vs. all other samples.

Article Snippet: . scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Incubation, Activity Assay, Immunoprecipitation, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Western blot analysis of α2M (A) and uPA (B) antigens in complexes immunoprecipitated from PFs of rabbits treated with intrapleural scuPA. Western blot analysis of proteins precipitated by anti-rabbit α2M (lanes 1–3) or nonspecific (lane 4) IgY from PFs of rabbits treated with ED scuPA. PFs were incubated with (lanes 2 and 4) or without (lanes 1 and 3) exogenous scuPA (50 nM) and precipitated with anti-rabbit α2M IgY as described in materials and methods and Fig. 5 legend. Samples of the resin were heated with SDS loading buffer (100°C, 2 min) and subjected to a 4–12% gradient SDS-PAGE (NuPage, Invitrogen) under nonreducing conditions. Positions of molecular weight markers (Novex, Invitrogen) are indicated to the left of the gel to allow assessment of the molecular weights of the bands. Lane 5: scuPA standard. Proteins were transferred to the PVDF membrane (Invitrogen), and membranes were incubated with anti-uPA MAb and developed with anti-human-α2M IgY-HRP conjugate (A) using ECL substrate (Pierce) and with goat anti-mouse IgG-alkaline phosphatase conjugate (B) using WesternBreeze (Invitrogen). Each set of 5 lanes (A and B) represents data obtained from the same gel.

Article Snippet: . scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Western Blot, Immunoprecipitation, Incubation, SDS Page, Molecular Weight, Membrane

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Rabbit α2M/uPA complexes immunoprecipitated from PFs activate plasminogen and increase uPA activity when exposed to plasmin. A: α2M complexes were isolated from PFs of animals treated with ED scuPA, high dose of Activase, Abbokinase, and with buffer saline vehicle by immunoprecipitation with anti-rabbit α2M IgY (gray) or nonspecific IgY (white). The resin was transferred into 96-well Pro-Bind plates and incubated with plasminogen (0.2 μM) and 0.2 mM chromogenic plasmin substrate (Centerchem, Switzerland) at 37°C. Plasminogen activation was determined as an increase in plasmin (PL) activity in time using SpectraMax Plus (Molecular Devices). The data are presented in the box plot format as described in Fig. 1 legend. Plasminogen activating activity was significantly increased in the PFs of rabbits treated with intrapleural scuPA (P < 0.05). B: effect of plasmin on uPA activity of α2M/uPA complexes precipitated with anti-rabbit α2M IgY from pleural fluids. PFs of animals treated with scuPA were incubated with or without a mixture of 50 nM scuPA and 100 nM PAI-1 in HEPES buffer, pH 7.4, for 5 h at 37°C. α2M/uPA complexes were precipitated with anti-rabbit IgY and Agarose with immobilized anti-IgY goat polyclonal IgG. Amidolytic uPA activity in the resin samples was measured with fluorogenic substrate Pefafluor uPA with (gray) or without (white) preincubation with 1 nM plasmin. *Significant differences (P < 0.05) between the groups.

Article Snippet: . scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) (30-100 nM) were incubated with either 100–250 nM suPAR (Attenuon) or with 0.5–2.5 μM α2M from human plasma (Meridian Life Science, Saco, ME) in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C.

Techniques: Immunoprecipitation, Activity Assay, Isolation, Saline, Incubation, Activation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of uPA with human plasma α2-macroglobulin (α2M), but not with human recombinant suPAR, results in protection from inactivation by PAI-1. scuPA (50 nM) was incubated with 150 nM suPAR, and 50 nM scuPA, HMW tcuPA, or Abbokinase (LMW tcuPA) were incubated with 2.0 μM α2M from human plasma in 0.05 M HEPES/NaOH buffer (pH 7.4) at 37°C for 7 h. At the end of incubation, complexes of uPA with suPAR or α2M were isolated from each reaction mixture by IP with 20 μg of MAb ATN-658 (ATN-658 does not affect amidolytic activity of uPA complexed with suPAR) and with 20 μg of polyclonal sheep anti-human α2M antibodies. Reaction mixtures were incubated with 40 μl of 1:1 slurry of protein A/G Plus Agarose for 2 h at room temperature on a rocker. Reaction mixtures were centrifuged at 3,000–6,000 rpm using an AccuSpin Micro R centrifuge. The resin was washed 3 times with 0.6 ml of cold HEPES buffer, and 50 μl of the resin suspension (total vol 200 μl) was transferred to 96-well plates. Amidolytic uPA activity in precipitated samples was measured with (white bars) or without (gray bars) 80 nM PAI-1. The composition of each reaction mixture, antibodies used for IP, and presence of PAI-1 are shown in the table under the plot.

Article Snippet: In control experiments, endogenous α2M/uPA complexes were precipitated from PFs using polyclonal sheep anti-human α2M antibodies (Meridian Life Science).

Techniques: Incubation, Clinical Proteomics, Recombinant, Isolation, Activity Assay, Suspension

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Incubation of scuPA with rabbit PFs forms αM complexes possessing uPA amidolytic activity that resists PAI-1. A: IP of uPA activity by anti-rabbit α2M IgY from PFs of rabbits treated with intrapleural ED of scuPA after preincubation with 50 nM scuPA for 5 h at 37°C in 50 mM HEPES/NaOH, pH 7.4. Samples were immunoprecipitated with α2M IgY or control nonspecific IgY, and uPA amidolytic activity was measured with (white) or without (gray) added exogenous PAI-1 (80 nM) as described in materials and methods and in Fig. 5. uPA amidolytic activity was significantly (*P < 0.05) increased in both groups of PFs from rabbits IP with anti-rabbit α2M vs. those IP nonspecific IgY. B: bar plot of the uPA activity in the precipitates from PFs of animals treated with the ED of scuPA or HD of Activase (Act), Abbokinase (Abb), or vehicle (n = 2 randomly selected samples/each paired group). The precipitation was carried out with 40 μg of either anti-rabbit α2M (filled bars) or nonspecific IgY (hatched bars). The complexes were captured with 80 μl of Agarose slurry and analyzed as described in Fig. 5. C: anti-α2M but not anti-uPAR antibodies precipitate uPA activity from PFs of rabbits, treated with ED of scuPA. IP of uPA activity by polyclonal sheep anti-human α2M antibodies and anti-human uPAR MAb (cross-reacting with rabbit αM and rabbit uPAR, respectively) from PFs of rabbits treated with ED of scuPA. PFs were incubated with 40 μg of antibodies for 2 h in 50 mM HEPES/NaOH, pH 7.4, either with anti-uPAR MAb or with anti-α2M antibodies. In control experiments, PFs were supplemented with exogenous mixtures of 150 nM suPAR with either 50 nM HMW or LMW tcuPA before anti-uPAR MAb was added. Another set of controls contained PFs without addition of antibodies. The composition of each reaction mixture and antibodies used for IP are shown in the table under the plot. Complexes were precipitated with 40 μl of Protein A/G Plus Agarose, and uPA amidolytic activity was measured with (white) or without (gray) 80 nM PAI-1 added, as described in Fig. 5 legend. uPA amidolytic activity was significantly (P < 0.05) increased in PFs incubated with sheep anti-human α2M antibodies and samples supplemented with HMW tcuPA complexed with suPAR (activity without PAI-1) vs. all other samples.

Article Snippet: In control experiments, endogenous α2M/uPA complexes were precipitated from PFs using polyclonal sheep anti-human α2M antibodies (Meridian Life Science).

Techniques: Incubation, Activity Assay, Immunoprecipitation, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Western blot analysis of α2M (A) and uPA (B) antigens in complexes immunoprecipitated from PFs of rabbits treated with intrapleural scuPA. Western blot analysis of proteins precipitated by anti-rabbit α2M (lanes 1–3) or nonspecific (lane 4) IgY from PFs of rabbits treated with ED scuPA. PFs were incubated with (lanes 2 and 4) or without (lanes 1 and 3) exogenous scuPA (50 nM) and precipitated with anti-rabbit α2M IgY as described in materials and methods and Fig. 5 legend. Samples of the resin were heated with SDS loading buffer (100°C, 2 min) and subjected to a 4–12% gradient SDS-PAGE (NuPage, Invitrogen) under nonreducing conditions. Positions of molecular weight markers (Novex, Invitrogen) are indicated to the left of the gel to allow assessment of the molecular weights of the bands. Lane 5: scuPA standard. Proteins were transferred to the PVDF membrane (Invitrogen), and membranes were incubated with anti-uPA MAb and developed with anti-human-α2M IgY-HRP conjugate (A) using ECL substrate (Pierce) and with goat anti-mouse IgG-alkaline phosphatase conjugate (B) using WesternBreeze (Invitrogen). Each set of 5 lanes (A and B) represents data obtained from the same gel.

Article Snippet: In control experiments, endogenous α2M/uPA complexes were precipitated from PFs using polyclonal sheep anti-human α2M antibodies (Meridian Life Science).

Techniques: Western Blot, Immunoprecipitation, Incubation, SDS Page, Molecular Weight, Membrane

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

doi: 10.1152/ajplung.00066.2009

Figure Lengend Snippet: Rabbit α2M/uPA complexes immunoprecipitated from PFs activate plasminogen and increase uPA activity when exposed to plasmin. A: α2M complexes were isolated from PFs of animals treated with ED scuPA, high dose of Activase, Abbokinase, and with buffer saline vehicle by immunoprecipitation with anti-rabbit α2M IgY (gray) or nonspecific IgY (white). The resin was transferred into 96-well Pro-Bind plates and incubated with plasminogen (0.2 μM) and 0.2 mM chromogenic plasmin substrate (Centerchem, Switzerland) at 37°C. Plasminogen activation was determined as an increase in plasmin (PL) activity in time using SpectraMax Plus (Molecular Devices). The data are presented in the box plot format as described in Fig. 1 legend. Plasminogen activating activity was significantly increased in the PFs of rabbits treated with intrapleural scuPA (P < 0.05). B: effect of plasmin on uPA activity of α2M/uPA complexes precipitated with anti-rabbit α2M IgY from pleural fluids. PFs of animals treated with scuPA were incubated with or without a mixture of 50 nM scuPA and 100 nM PAI-1 in HEPES buffer, pH 7.4, for 5 h at 37°C. α2M/uPA complexes were precipitated with anti-rabbit IgY and Agarose with immobilized anti-IgY goat polyclonal IgG. Amidolytic uPA activity in the resin samples was measured with fluorogenic substrate Pefafluor uPA with (gray) or without (white) preincubation with 1 nM plasmin. *Significant differences (P < 0.05) between the groups.

Article Snippet: In control experiments, endogenous α2M/uPA complexes were precipitated from PFs using polyclonal sheep anti-human α2M antibodies (Meridian Life Science).

Techniques: Immunoprecipitation, Activity Assay, Isolation, Saline, Incubation, Activation Assay