10h10 antibody  (Bio-Rad)

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: HDBECs (2×10 5 ) were incubated for 24 h with recombinant TF (0, 0.5 and 2 U/ml) or PAR2-AP (SLIGKV; 20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to addition of TF. The cells were harvested after 24 h, into separate aliquots (1×10 5 cells). Total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) Inhibitor of CDK p16 INKa mRNA (n=5), and (B) the relative amounts of p16 INKa protein (n=3) calculated from (C) the western blots of p16 INKa protein (using a goat anti-human antibody) and against GAPDH. TF, tissue factor; PAR2, protease-activated receptor 2.

Article Snippet: In some experiments, TF was pre-incubated at 37°C for 60 min with 10H10 antibody (20 μg/ml; cat. no. 9010-5059; Bio-Rad Laboratories, Inc.) to block TF proliferative signalling via the TF exosite ( ) or HTF1 antibody (20 μg/ml; cat. no. 16-1429-85; eBioscience; Thermo Fisher Scientific, Inc.) to block the protease activity of the TF-fVIIa complex ( ).

Techniques: Incubation, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: HDBECs (2×10 5 ) were incubated for 24 h with recombinant TF (0, 0.5 and 2 U/ml) or PAR2-AP (SLIGKV; 20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Groups of cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to the addition of TF. The cells were harvested after 24 h, into separate aliquots (1×10 5 cells). Total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 mRNA (n=5), and (B) the relative amounts of p21 CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21 CIP1/WAF1 protein, (using a mouse anti-human antibody) and against GAPDH. TF, tissue factor; PAR2, protease-activated receptor 2.

Article Snippet: In some experiments, TF was pre-incubated at 37°C for 60 min with 10H10 antibody (20 μg/ml; cat. no. 9010-5059; Bio-Rad Laboratories, Inc.) to block TF proliferative signalling via the TF exosite ( ) or HTF1 antibody (20 μg/ml; cat. no. 16-1429-85; eBioscience; Thermo Fisher Scientific, Inc.) to block the protease activity of the TF-fVIIa complex ( ).

Techniques: Incubation, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Examination of the influence of TF on cell cycle indicators. (A) Groups of HDBECs (1×10 5 ) were transfected with the pGL3-promoter vector containing the E2F-1 enhancer sequence. The cells were then treated for 24 h with recombinant TF (0, 0.5 and 2 U/ml), combinations of TF (0.5 U/ml) with the shown antibodies, or with PAR2-AP (SLIGKV; 20 µM) and the luciferase activity was measured within 24 h (n=3). (B) Groups of cells (5×10 4 ) were seeded in 96-well plates and treated with recombinant TF (0, 0.5 and 2 U/ml) for 24 h. The cells were then fixed and probed with an anti-phospho-Thr821/826 human retinoblastoma protein antibody (1:1,000 v/v) for 1 h. The samples were then incubated with an HRP-conjugated donkey anti-goat IgG diluted 1:3,000 v/v) for 1 h, developed with a TMB substrate and the absorptions determined at 450 nm (n=3). (C) Groups of cells (1×10 5 ) were incubated for 24 h with TF (0, 0.5 and 2 U/ml) or PAR2-AP (20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Groups of cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to addition of recombinant TF. The cells were harvested after 24 h, total RNA was isolated and the amount of cyclin D1 mRNA determined against that of β-actin (n=4). (D) Groups of cells (1×10 5 ) were treated as aforementioned and cell numbers were determined using crystal violet staining (n=3). TF, tissue factor; PAR2, protease-activated receptor 2; E2F-1; Early region 2 binding factor.

Article Snippet: In some experiments, TF was pre-incubated at 37°C for 60 min with 10H10 antibody (20 μg/ml; cat. no. 9010-5059; Bio-Rad Laboratories, Inc.) to block TF proliferative signalling via the TF exosite ( ) or HTF1 antibody (20 μg/ml; cat. no. 16-1429-85; eBioscience; Thermo Fisher Scientific, Inc.) to block the protease activity of the TF-fVIIa complex ( ).

Techniques: Transfection, Plasmid Preparation, Sequencing, Recombinant, Luciferase, Activity Assay, Incubation, Isolation, Staining, Binding Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial-derived microvesicles promote pro-migratory cross-talk with smooth muscle cells by a mechanism requiring tissue factor and PAR2 activation

doi: 10.3389/fcvm.2024.1365008

Figure Lengend Snippet: Examination of the pro-migratory mechanism of TF-MV using blocking antibodies. MV were prepared from HCAEC (5 × 10 5 ) transfected to express TF Wt -tGFP and pre-incubated with mouse anti-human-TF antibodies, 10H10 (20 µg/ml) or HTF1 (20 µg/ml), or an inhibitory polyclonal rabbit anti-human fVIIa antibody (10 µg/ml). Additionally, HCASMC were incubated with an inhibitory mouse anti-human PAR2 antibody, SAM11 (20 µg/ml). HCASMC (3 × 10 4 ) were stimulated with MV at 37°C for 18 h. The cells were then fixed, washed and the cells on the upper side of the chamber were scraped off. ( A ) The cells were stained with crystal violet and photographed and ( B ) the numbers of migrating cells were determined by eluting the crystal violet and measuring the absorptions at 595 nm. Presented data include the calculated mean values ± the calculated standard error of the mean, from 5 experiments each examined in duplicate.

Article Snippet: In some experiments, the isolated MV were pre-incubated for 1 h, with a mouse anti-human-TF antibody, 10H10 (20 µg/ml; BD Bioscience, Wokingham, UK) capable of blocking TF signaling, a mouse anti-human-TF antibody, HTF-1 (20 µg/ml; eBioscience/Thermo Scientific, Warrington, UK) to block TF-fVIIa protease/procoagulant activity, an inhibitory polyclonal rabbit anti-human fVIIa antibody (10 µg/ml; Abcam, Cambridge, UK) or the respective control isotype IgG antibodies (20 µg/ml; New England Biolabs, Hitchin, UK).

Techniques: Blocking Assay, Transfection, Incubation, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Endothelial-derived microvesicles promote pro-migratory cross-talk with smooth muscle cells by a mechanism requiring tissue factor and PAR2 activation

doi: 10.3389/fcvm.2024.1365008

Figure Lengend Snippet: Examination of the pro-migratory mechanism of TF-MV using blocking antibodies. MV were prepared from HCAEC (5 × 10 5 ) transfected to express TF Wt -tGFP and pre-incubated with mouse anti-human-TF antibodies, 10H10 (20 µg/ml) or HTF1 (20 µg/ml), or an inhibitory polyclonal rabbit anti-human fVIIa antibody (10 µg/ml). Additionally, HCASMC were incubated with an inhibitory mouse anti-human PAR2 antibody, SAM11 (20 µg/ml). HCASMC (3 × 10 4 ) were stimulated with MV at 37°C for 18 h. The cells were then fixed, washed and the cells on the upper side of the chamber were scraped off. ( A ) The cells were stained with crystal violet and photographed and ( B ) the numbers of migrating cells were determined by eluting the crystal violet and measuring the absorptions at 595 nm. Presented data include the calculated mean values ± the calculated standard error of the mean, from 5 experiments each examined in duplicate.

Article Snippet: In addition, the association of TF-tGFP with the c-terminal of filamin A was confirmed in situ by proximity ligation assay as before ( , ), and using a rabbit monoclonal antibody against the c-terminal of filamin A (EP2405Y), in conjunction with first a mouse monoclonal antibody to TF (10H10) and also, with a mouse monoclonal antibody to tGFP protein (2HB, OriGene) ( ).

Techniques: Blocking Assay, Transfection, Incubation, Staining