Figure 2.
TF-FVIIa triggers a distinct PAR2 signaling pathway that involves PI3 kinase–dependent MAP kinase signaling. (A) IL-8 mRNA quantification of HaCaT cells stimulated with FVIIa (10 nM) and PAR2 agonist SLIGRL (50 μM) with or without MEK inhibitor U0126 (1 μM) and PI3K inhibitor LY294002 (1 μM) (n = 3-5). ***P < .001, one-way ANOVA and Dunnett’s test. (B) Time course of ERK phosphorylation (pERK) in HaCaT cells stimulated with FVIIa (10 nM) or SLIGRL (50 μM). Upper panel: representative western blots of pERK and total ERK. Lower panel: densitometric quantification of pERK western blots from 3 independent experiments (n = 7). *P < .05, Student t test. (C) Western blots for pERK levels in HaCaT cells stimulated (stim) for 30 minutes with FVIIa (10 nM) and SLIGRL (50 μM) in the absence or presence of MEK inhibitor U0126 (1 μM) and PI3 kinase inhibitor LY294002 (1 μM). (D) Western blots of TF and PAR2 in A7 cells 24 and 48 hours after adenoviral transduction to express PAR2 as well as TF and TFΔCT, respectively. (E) Quantification of IL-8 mRNA in A7 cells that were transduced to express TF wt and PAR2 48 hours before the experiment and stimulated with FVIIa (10 nM) for 90 minutes with and without MEK inhibitor U0126 (1 μM) or PI3 kinase inhibitor LY294002 (1 μM). (F) Quantification of IL-8 mRNA in A7 cells expressing full-length TF or TFΔCT stimulated for 90 minutes with FVIIa (10 nM) or SLIGRL (50 μM) (n = 3). *P < .05, paired Student t test. (G) Western blot of TF and p55 in TF immunoprecipitates using αTF 5G9 antibody coupled to DynaBeads from A7 melanoma cells expressing full-length TF or TFΔCT. (H) Representative western blots and densitometric quantification of 4 independent experiments of TF in a TS2/16 pull-down assay and cleared cell lysates from A7 cells expressing TF wt and TFΔCT, respectively, after 30 minutes of stimulation with FVIIa (10 nM) (n = 4). *P < .05, one-way ANOVA and Tukey’s test. CL, cleared-cell lysate; IP, immunoprecipitate.

TF-FVIIa triggers a distinct PAR2 signaling pathway that involves PI3 kinase–dependent MAP kinase signaling. (A) IL-8 mRNA quantification of HaCaT cells stimulated with FVIIa (10 nM) and PAR2 agonist SLIGRL (50 μM) with or without MEK inhibitor U0126 (1 μM) and PI3K inhibitor LY294002 (1 μM) (n = 3-5). ***P < .001, one-way ANOVA and Dunnett’s test. (B) Time course of ERK phosphorylation (pERK) in HaCaT cells stimulated with FVIIa (10 nM) or SLIGRL (50 μM). Upper panel: representative western blots of pERK and total ERK. Lower panel: densitometric quantification of pERK western blots from 3 independent experiments (n = 7). *P < .05, Student t test. (C) Western blots for pERK levels in HaCaT cells stimulated (stim) for 30 minutes with FVIIa (10 nM) and SLIGRL (50 μM) in the absence or presence of MEK inhibitor U0126 (1 μM) and PI3 kinase inhibitor LY294002 (1 μM). (D) Western blots of TF and PAR2 in A7 cells 24 and 48 hours after adenoviral transduction to express PAR2 as well as TF and TFΔCT, respectively. (E) Quantification of IL-8 mRNA in A7 cells that were transduced to express TF wt and PAR2 48 hours before the experiment and stimulated with FVIIa (10 nM) for 90 minutes with and without MEK inhibitor U0126 (1 μM) or PI3 kinase inhibitor LY294002 (1 μM). (F) Quantification of IL-8 mRNA in A7 cells expressing full-length TF or TFΔCT stimulated for 90 minutes with FVIIa (10 nM) or SLIGRL (50 μM) (n = 3). *P < .05, paired Student t test. (G) Western blot of TF and p55 in TF immunoprecipitates using αTF 5G9 antibody coupled to DynaBeads from A7 melanoma cells expressing full-length TF or TFΔCT. (H) Representative western blots and densitometric quantification of 4 independent experiments of TF in a TS2/16 pull-down assay and cleared cell lysates from A7 cells expressing TF wt and TFΔCT, respectively, after 30 minutes of stimulation with FVIIa (10 nM) (n = 4). *P < .05, one-way ANOVA and Tukey’s test. CL, cleared-cell lysate; IP, immunoprecipitate.

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