Figure 3.
FVIIa promotes TF complex formation with the active conformer of integrin β1. (A) TF-integrin β1 interaction in HaCaT cells stimulated for indicated time points with FVIIa (10 nM). Left panel: representative western blots of TF and integrin β1 in immunoprecipitates from Brij 35 cell lysates using DynaBead-coupled integrin β1-specific antibodies TS2/16 (active conformer) or AIIB2. Right panel: densitometric quantification of western blots (n = 4). *P < .05, **P < .01, one-way ANOVA and Dunnett’s test. (B) Representative western blot of TF and integrin β1 in a TS2/16 pull-down assay of Brij 35 lysates from HaCaT cells stimulated with FVIIa (10 nM) and active-site inhibited FVIIai (10 nM) for 30 minutes. (C) Quantification of IL-8 mRNA in FVIIa-stimulated cells (10 nM) in the absence or presence of αTF antibody 10H10 (50 μg/mL) or control immunoglobulin G TIB115 (50 μg/mL) (n = 3). **P < .01, one-way ANOVA and Dunnett’s test. (D) FVIIa-induced TF-integrin β1 interaction in HaCaT cells incubated with or without 10H10 (50 μg/mL). Left panel: representative western blots of TF and integrin β1 in TS2/16 immunoprecipitates. Right panel: densitometric quantification of western blots (n = 4). ***P < .01, Student t test. (E) Quantification of IL-8 mRNA in MDA-MB-231 cells stimulated for 90 minutes with recombinant matriptase (rhST-14, 5 nM), FVIIa (10 nM), or FVIIa (0.5 nM)/FX (50 nM) in the absence or presence of serine protease inhibitor aprotinin (5 μM) (n = 3). *P < .05, Student t test. (F) Representative western blot of TF in a TS2/16- and AIIB2 pull-down assay of Brij 35 lysates from MDA-MB-231 cells stimulated with FVIIa (10 nM) for 30 minutes. (G) Representative western blots of TF and integrin β1 in immunoprecipitates from Brij 35 cell lysates using DynaBead-coupled FVIIa-specific antibody 12C7. (H) For intracellular tracking of FVIIa, fluorophore-conjugate antibody anti-FVIIa 12C7-Alexa647 (5 μg/mL) was added to FVIIa stimulation (10 nM for indicated time) in MDA-MB-231 cells. After fixation, cells were counterstained for integrin β1 with TS2/16-fluorescein isothiocyanate (FITC) (5 μg/mL) and nuclei (Hoechst, 1 μg/mL). Scale bar = 10 μm. (I) Similarly, FVIIa was tracked in HaCaT cells over 30 minutes of stimulation by using fluorophore-conjugate antibody anti-FVIIa 12C7-Alexa647 (5 μg/mL) and counterstaining for integrin β1 with TS2/16-FITC. Scale bar = 10 μm. (J) Postfixation staining of MDA-MB-231 cells after incubation with FVIIa for 5 and 15 minutes with anti-FVIIa 3G12-Alexa647 with and without permeabilization after blockade of surface FVIIa with unlabeled antibody. Confocal images of (K) MDA-MB-231 cells and (L) HaCaT cells incubated with anti-FVIIa 12C7-Alexa647 (5 μg/mL) during 30 minutes of stimulation with FVIIa (10 nM). Cells were counterstained for early endosomal marker EEA1 (αEEA1-Alexa488 conjugate, 5 μg/mL) and nuclei (Hoechst, 1 μg/mL) postfixation. Scale bar = 10 μm. con, control.

FVIIa promotes TF complex formation with the active conformer of integrin β1. (A) TF-integrin β1 interaction in HaCaT cells stimulated for indicated time points with FVIIa (10 nM). Left panel: representative western blots of TF and integrin β1 in immunoprecipitates from Brij 35 cell lysates using DynaBead-coupled integrin β1-specific antibodies TS2/16 (active conformer) or AIIB2. Right panel: densitometric quantification of western blots (n = 4). *P < .05, **P < .01, one-way ANOVA and Dunnett’s test. (B) Representative western blot of TF and integrin β1 in a TS2/16 pull-down assay of Brij 35 lysates from HaCaT cells stimulated with FVIIa (10 nM) and active-site inhibited FVIIai (10 nM) for 30 minutes. (C) Quantification of IL-8 mRNA in FVIIa-stimulated cells (10 nM) in the absence or presence of αTF antibody 10H10 (50 μg/mL) or control immunoglobulin G TIB115 (50 μg/mL) (n = 3). **P < .01, one-way ANOVA and Dunnett’s test. (D) FVIIa-induced TF-integrin β1 interaction in HaCaT cells incubated with or without 10H10 (50 μg/mL). Left panel: representative western blots of TF and integrin β1 in TS2/16 immunoprecipitates. Right panel: densitometric quantification of western blots (n = 4). ***P < .01, Student t test. (E) Quantification of IL-8 mRNA in MDA-MB-231 cells stimulated for 90 minutes with recombinant matriptase (rhST-14, 5 nM), FVIIa (10 nM), or FVIIa (0.5 nM)/FX (50 nM) in the absence or presence of serine protease inhibitor aprotinin (5 μM) (n = 3). *P < .05, Student t test. (F) Representative western blot of TF in a TS2/16- and AIIB2 pull-down assay of Brij 35 lysates from MDA-MB-231 cells stimulated with FVIIa (10 nM) for 30 minutes. (G) Representative western blots of TF and integrin β1 in immunoprecipitates from Brij 35 cell lysates using DynaBead-coupled FVIIa-specific antibody 12C7. (H) For intracellular tracking of FVIIa, fluorophore-conjugate antibody anti-FVIIa 12C7-Alexa647 (5 μg/mL) was added to FVIIa stimulation (10 nM for indicated time) in MDA-MB-231 cells. After fixation, cells were counterstained for integrin β1 with TS2/16-fluorescein isothiocyanate (FITC) (5 μg/mL) and nuclei (Hoechst, 1 μg/mL). Scale bar = 10 μm. (I) Similarly, FVIIa was tracked in HaCaT cells over 30 minutes of stimulation by using fluorophore-conjugate antibody anti-FVIIa 12C7-Alexa647 (5 μg/mL) and counterstaining for integrin β1 with TS2/16-FITC. Scale bar = 10 μm. (J) Postfixation staining of MDA-MB-231 cells after incubation with FVIIa for 5 and 15 minutes with anti-FVIIa 3G12-Alexa647 with and without permeabilization after blockade of surface FVIIa with unlabeled antibody. Confocal images of (K) MDA-MB-231 cells and (L) HaCaT cells incubated with anti-FVIIa 12C7-Alexa647 (5 μg/mL) during 30 minutes of stimulation with FVIIa (10 nM). Cells were counterstained for early endosomal marker EEA1 (αEEA1-Alexa488 conjugate, 5 μg/mL) and nuclei (Hoechst, 1 μg/mL) postfixation. Scale bar = 10 μm. con, control.

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