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monoclonal mouse anti-magi-1 (Novus Biologicals)

Name: monoclonal mouse anti-magi-1
Brand: Novus Biologicals
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Novus Biologicals monoclonal mouse anti-magi-1
Monoclonal Mouse Anti Magi 1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews

monoclonal mouse anti-magi-1 - by Bioz Stars, 2026-03

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Fig. 1 Analysis of the interaction of TF and MAGI1-3 by proximity ligation assay and the influence of PAR2 activation. MDA-MB-231 cells (103) were seeded out into 35 mm-glass based μ-dishes and adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 30 min and then fixed with 4% (v/v) paraformaldehyde for 15 min. The cells were washed three times with PBS and permeabilised with Triton X-100 0.1% (v/v) in PBS, for 5 min. All samples were blocked with Duolink blocking buffer for 1 h and incubated overnight with combinations of antibodies as follows, at 4 °C. The proximity between TF and MAGI1-3 were examined using a mouse anti-TF antibody HTF1 (5 μg/ml) together with a rabbit anti-MAGI1 antibody (H-70; 2 μg/ml), a rabbit anti-MAGI2 antibody (2 μg/ml) or a rabbit anti-MAGI3 antibody (2 μg/ml). The antibodies were diluted in the provided antibody diluent and blocked with the provided blocking buffer. The cells were washed three times with PBS and PLA performed according to the manufacturer’s instructions. The cells were labelled with DAPI (2 μg/ml) and Phalloidin-FITC (2 µg/ml). Images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a × 40 magnification. (The micrographs are representative of 5 fields of view from 9 experiments, RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI). B The interactions of TF with MAGI1-3 in non-activated and at 20 min post-activation was analysed. C The interaction of TF and MAGI1 at intervals up to 40 min was analysed by PLA

Journal: Thrombosis journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1.

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Fig. 1 Analysis of the interaction of TF and MAGI1-3 by proximity ligation assay and the influence of PAR2 activation. MDA-MB-231 cells (103) were seeded out into 35 mm-glass based μ-dishes and adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 30 min and then fixed with 4% (v/v) paraformaldehyde for 15 min. The cells were washed three times with PBS and permeabilised with Triton X-100 0.1% (v/v) in PBS, for 5 min. All samples were blocked with Duolink blocking buffer for 1 h and incubated overnight with combinations of antibodies as follows, at 4 °C. The proximity between TF and MAGI1-3 were examined using a mouse anti-TF antibody HTF1 (5 μg/ml) together with a rabbit anti-MAGI1 antibody (H-70; 2 μg/ml), a rabbit anti-MAGI2 antibody (2 μg/ml) or a rabbit anti-MAGI3 antibody (2 μg/ml). The antibodies were diluted in the provided antibody diluent and blocked with the provided blocking buffer. The cells were washed three times with PBS and PLA performed according to the manufacturer’s instructions. The cells were labelled with DAPI (2 μg/ml) and Phalloidin-FITC (2 µg/ml). Images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a × 40 magnification. (The micrographs are representative of 5 fields of view from 9 experiments, RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI). B The interactions of TF with MAGI1-3 in non-activated and at 20 min post-activation was analysed. C The interaction of TF and MAGI1 at intervals up to 40 min was analysed by PLA

Article Snippet: In order to assess the proximity and potential interaction between TF and MAGI1-3, a mouse anti-TF antibody (HTF1; 5 μg/ ml; eBioscience/Thermo Scientific, Warrington, UK) was used together with a rabbit anti-MAGI1 antibody (H-70; 2 μg/ml; Santa Cruz Biotechnology, Heidelberg, Germany), a rabbit anti-MAGI2 antibody (2 μg/ml; GenTex/Insight Biotechnologies, Wembley, UK) or a rabbit anti-MAGI3 antibody (2 μg/ml; Novus/R&D Systems, Abingdon, UK).

Techniques: Proximity Ligation Assay, Activation Assay, Incubation, Blocking Assay, Fluorescence, Microscopy

Fig. 2 Analysis of the interaction of TF and MAGI1-3 and the influence of PAR2 activation. A MDA-MB-231 cells (2 × 105) were adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 20 min and TF was immunoprecipitated from cell lysates with the anti-TF (HTF-1; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for MAGI1 and TF by western blot using an anti-MAGI1 (H-70) and a rabbit anti-TF antibody (FL-295). B The ratio of the MAGI1 band densities were normalised against those of TF in the same co-immunoprecipitated samples. C In addition, MAGI1 was immunoprecipitated from cell lysates with an anti-MAGI1 (H-70; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for TF and MAGI1 by western blot using an anti-TF antibody (HTF-1) and a mouse anti-MAGI1 antibody (SS-5). D The ratios of the TF band densities were normalised against those of MAGI1 in the same co-immunoprecipitated samples

Journal: Thrombosis journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1.

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Fig. 2 Analysis of the interaction of TF and MAGI1-3 and the influence of PAR2 activation. A MDA-MB-231 cells (2 × 105) were adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 20 min and TF was immunoprecipitated from cell lysates with the anti-TF (HTF-1; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for MAGI1 and TF by western blot using an anti-MAGI1 (H-70) and a rabbit anti-TF antibody (FL-295). B The ratio of the MAGI1 band densities were normalised against those of TF in the same co-immunoprecipitated samples. C In addition, MAGI1 was immunoprecipitated from cell lysates with an anti-MAGI1 (H-70; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for TF and MAGI1 by western blot using an anti-TF antibody (HTF-1) and a mouse anti-MAGI1 antibody (SS-5). D The ratios of the TF band densities were normalised against those of MAGI1 in the same co-immunoprecipitated samples

Techniques: Activation Assay, Incubation, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot

Fig. 3 Qualitative co-localisation of TF and MAGI1 in resting and activated MDA-MB-231 cells. MDA-MB-231 cells (104) were seeded out into 35 mm-glass based μ-dishes and sets were activated by incubation with PAR2-AP (20 μM). The cells were fixed and probed for TF using an FITC-conjugated anti-TF antibody (HTF1-FITC). The patterns were compared to MAGI1 probed with a rabbit anti-MAGI1 antibody (H-70) and developed a goat anti-rabbit IgG-AlexaFluor 594 antibody. The cells were also stained with DAPI (2 μg/ml) and examined by fluorescence microscopy. Co-localisation coefficient values were then determiend using the ImageJ program. The values were calculated as the average from 10 captured images, and the data show the values for 5 separate experiments

Journal: Thrombosis journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1.

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Fig. 3 Qualitative co-localisation of TF and MAGI1 in resting and activated MDA-MB-231 cells. MDA-MB-231 cells (104) were seeded out into 35 mm-glass based μ-dishes and sets were activated by incubation with PAR2-AP (20 μM). The cells were fixed and probed for TF using an FITC-conjugated anti-TF antibody (HTF1-FITC). The patterns were compared to MAGI1 probed with a rabbit anti-MAGI1 antibody (H-70) and developed a goat anti-rabbit IgG-AlexaFluor 594 antibody. The cells were also stained with DAPI (2 μg/ml) and examined by fluorescence microscopy. Co-localisation coefficient values were then determiend using the ImageJ program. The values were calculated as the average from 10 captured images, and the data show the values for 5 separate experiments

Techniques: Incubation, Staining, Fluorescence, Microscopy

Fig. 4 Examination of influence of TF phosphorylation on the binding of MAGI1 and 3. Biotinylated peptides, corresponding to the last 18 amino acids of the cytoplasmic domain of TF were synthesised in non-phosphorylated, single-phosphorylated and double-phosphorylated forms and used as bait. An additional scrambled peptide (biotin-SWGNVSKLSAPRQGVNKE) was also included alongside. The peptides (5 µM final concentration) in PBS, were bound into a NeutrAvidin-coated 96-well plate and then blocked. Cell lysates from resting and PAR2-activated MDA-MB-231 cells (from 2 × 105 cells) were incubated in the plates for 1 h at room temperature. The wells were then washed four times and probed with (A) rabbit anti-MAGI1 (H-70) or (B) mouse anti-MAGI3 (46) antibodies diluted 1:200 (v/v) in PBST. The samples were detected using goat anti-rabbit and goat anti-mouse alkaline phosphatase-conjugated antibodies diluted 1:200 (v/v) and the colour developed with TMB One Solution (100 µl). Once the colour was developed the reactions were stopped and absorptions recorded. (n = 3; * = p < 0.05 vs. the respective samples without cell lysate). C The interaction of MAGI1 with the non-phosphorylated TF peptide was examined by pull-down over a period of 30 min following activation of PAR2, as described above

Journal: Thrombosis journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1.

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Fig. 4 Examination of influence of TF phosphorylation on the binding of MAGI1 and 3. Biotinylated peptides, corresponding to the last 18 amino acids of the cytoplasmic domain of TF were synthesised in non-phosphorylated, single-phosphorylated and double-phosphorylated forms and used as bait. An additional scrambled peptide (biotin-SWGNVSKLSAPRQGVNKE) was also included alongside. The peptides (5 µM final concentration) in PBS, were bound into a NeutrAvidin-coated 96-well plate and then blocked. Cell lysates from resting and PAR2-activated MDA-MB-231 cells (from 2 × 105 cells) were incubated in the plates for 1 h at room temperature. The wells were then washed four times and probed with (A) rabbit anti-MAGI1 (H-70) or (B) mouse anti-MAGI3 (46) antibodies diluted 1:200 (v/v) in PBST. The samples were detected using goat anti-rabbit and goat anti-mouse alkaline phosphatase-conjugated antibodies diluted 1:200 (v/v) and the colour developed with TMB One Solution (100 µl). Once the colour was developed the reactions were stopped and absorptions recorded. (n = 3; * = p < 0.05 vs. the respective samples without cell lysate). C The interaction of MAGI1 with the non-phosphorylated TF peptide was examined by pull-down over a period of 30 min following activation of PAR2, as described above

Techniques: Phospho-proteomics, Binding Assay, Concentration Assay, Incubation, Activation Assay

Fig. 5 Identification of the PDZ domain within MAGI1 responsible for binding TF. MDA-MB-231 cells (103) were seeded out into 35 mm-glass based μ-dishes and separately transfected with constructs to express PDZ1-5 of from MAGI1. The cells permitted to express the proteins for 24 h and then fixed and washed. A The proximity of the expressed PDZ to TF was examined by PLA using a rabbit anti-HA (C2954) antibody and a mouse anti-TF (HTF1) antibody, respectively (RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI) and (B) quantified using the ImageJ program. C Cell surface TF-fVIIa activity was measured on the transfected cells expressing PDZ1, PDZ2 or the empty vector, using a modified thrombin-generation assay. An additional sample of cells were transfected to express PDZ1 but were pre-incubated with an inhibitory anti-TF antibody (HTF1; 20 µg/ ml) before analysis. The cells (105) were incubated for 20 min at 37 °C with a mixture of barium sulphate absorbable proteins (2 mg/ml) and 5 mM CaCl2 in the reaction buffer (total volume 150 µl; Tris-buffered saline pH 7.4), containing 1% (w/v) bovine serum albumin (BSA). Aliquots of reaction (100 µl) were then transferred to 96-well plates containing 100 µl of thrombin substrate CS-01(38) (0.2 µM H–D-Phe-Pip-Arg-pNA) and incubated for a further 40 min at 37 °C. The reactions were stopped by the addition of 2% (v/v) acetic acid (50 µl) and the absorption of the sample measured at 410 nm on a plate reader

Journal: Thrombosis journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1.

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Fig. 5 Identification of the PDZ domain within MAGI1 responsible for binding TF. MDA-MB-231 cells (103) were seeded out into 35 mm-glass based μ-dishes and separately transfected with constructs to express PDZ1-5 of from MAGI1. The cells permitted to express the proteins for 24 h and then fixed and washed. A The proximity of the expressed PDZ to TF was examined by PLA using a rabbit anti-HA (C2954) antibody and a mouse anti-TF (HTF1) antibody, respectively (RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI) and (B) quantified using the ImageJ program. C Cell surface TF-fVIIa activity was measured on the transfected cells expressing PDZ1, PDZ2 or the empty vector, using a modified thrombin-generation assay. An additional sample of cells were transfected to express PDZ1 but were pre-incubated with an inhibitory anti-TF antibody (HTF1; 20 µg/ ml) before analysis. The cells (105) were incubated for 20 min at 37 °C with a mixture of barium sulphate absorbable proteins (2 mg/ml) and 5 mM CaCl2 in the reaction buffer (total volume 150 µl; Tris-buffered saline pH 7.4), containing 1% (w/v) bovine serum albumin (BSA). Aliquots of reaction (100 µl) were then transferred to 96-well plates containing 100 µl of thrombin substrate CS-01(38) (0.2 µM H–D-Phe-Pip-Arg-pNA) and incubated for a further 40 min at 37 °C. The reactions were stopped by the addition of 2% (v/v) acetic acid (50 µl) and the absorption of the sample measured at 410 nm on a plate reader

Techniques: Binding Assay, Transfection, Construct, Activity Assay, Expressing, Plasmid Preparation, Modification, Incubation, Saline

Fig. 8 Schematic representation of the regulation of TF activity by interaction with MAGI1. A The interaction of the cytoplasmic domain of TF with the PDZ1 domain of MAGI1 restrains TF. Initiation of cell signalling following the activation of PAR2 on the cell surface may result in (B) the induction of capsase-1 leading to the degradation on MAGI1 into non-functional fragments. C Concurrent phosphorylation of TF by PKC at Ser253 prevents further binding with other MAGI1 proteins on the cells surface. These alterations collectively may promote the processing of TF permitting interaction with fVIIa as an active complex

Journal: Thrombosis journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1.

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Fig. 8 Schematic representation of the regulation of TF activity by interaction with MAGI1. A The interaction of the cytoplasmic domain of TF with the PDZ1 domain of MAGI1 restrains TF. Initiation of cell signalling following the activation of PAR2 on the cell surface may result in (B) the induction of capsase-1 leading to the degradation on MAGI1 into non-functional fragments. C Concurrent phosphorylation of TF by PKC at Ser253 prevents further binding with other MAGI1 proteins on the cells surface. These alterations collectively may promote the processing of TF permitting interaction with fVIIa as an active complex

Techniques: Activity Assay, Activation Assay, Functional Assay, Phospho-proteomics, Binding Assay

Analysis of the interaction of TF and MAGI1-3 by proximity ligation assay and the influence of PAR2 activation. MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes and adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 30 min and then fixed with 4% (v/v) paraformaldehyde for 15 min. The cells were washed three times with PBS and permeabilised with Triton X-100 0.1% (v/v) in PBS, for 5 min. All samples were blocked with Duolink blocking buffer for 1 h and incubated overnight with combinations of antibodies as follows, at 4 °C. The proximity between TF and MAGI1-3 were examined using a mouse anti-TF antibody HTF1 (5 μg/ml) together with a rabbit anti-MAGI1 antibody (H-70; 2 μg/ml), a rabbit anti-MAGI2 antibody (2 μg/ml) or a rabbit anti-MAGI3 antibody (2 μg/ml). The antibodies were diluted in the provided antibody diluent and blocked with the provided blocking buffer. The cells were washed three times with PBS and PLA performed according to the manufacturer’s instructions. The cells were labelled with DAPI (2 μg/ml) and Phalloidin-FITC (2 µg/ml). Images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a × 40 magnification. (The micrographs are representative of 5 fields of view from 9 experiments, RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI). B The interactions of TF with MAGI1-3 in non-activated and at 20 min post-activation was analysed. C The interaction of TF and MAGI1 at intervals up to 40 min was analysed by PLA

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Analysis of the interaction of TF and MAGI1-3 by proximity ligation assay and the influence of PAR2 activation. MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes and adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 30 min and then fixed with 4% (v/v) paraformaldehyde for 15 min. The cells were washed three times with PBS and permeabilised with Triton X-100 0.1% (v/v) in PBS, for 5 min. All samples were blocked with Duolink blocking buffer for 1 h and incubated overnight with combinations of antibodies as follows, at 4 °C. The proximity between TF and MAGI1-3 were examined using a mouse anti-TF antibody HTF1 (5 μg/ml) together with a rabbit anti-MAGI1 antibody (H-70; 2 μg/ml), a rabbit anti-MAGI2 antibody (2 μg/ml) or a rabbit anti-MAGI3 antibody (2 μg/ml). The antibodies were diluted in the provided antibody diluent and blocked with the provided blocking buffer. The cells were washed three times with PBS and PLA performed according to the manufacturer’s instructions. The cells were labelled with DAPI (2 μg/ml) and Phalloidin-FITC (2 µg/ml). Images were acquired using a Zeiss Axio Vert.A1 inverted fluorescence microscope with a × 40 magnification. (The micrographs are representative of 5 fields of view from 9 experiments, RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI). B The interactions of TF with MAGI1-3 in non-activated and at 20 min post-activation was analysed. C The interaction of TF and MAGI1 at intervals up to 40 min was analysed by PLA

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Proximity Ligation Assay, Activation Assay, Incubation, Blocking Assay, Fluorescence, Microscopy

Qualitative co-localisation of TF and MAGI1 in resting and activated MDA-MB-231 cells . MDA-MB-231 cells (10 4 ) were seeded out into 35 mm-glass based μ-dishes and sets were activated by incubation with PAR2-AP (20 μM). The cells were fixed and probed for TF using an FITC-conjugated anti-TF antibody (HTF1-FITC). The patterns were compared to MAGI1 probed with a rabbit anti-MAGI1 antibody (H-70) and developed a goat anti-rabbit IgG-AlexaFluor 594 antibody. The cells were also stained with DAPI (2 μg/ml) and examined by fluorescence microscopy. Co-localisation coefficient values were then determiend using the ImageJ program. The values were calculated as the average from 10 captured images, and the data show the values for 5 separate experiments

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Qualitative co-localisation of TF and MAGI1 in resting and activated MDA-MB-231 cells . MDA-MB-231 cells (10 4 ) were seeded out into 35 mm-glass based μ-dishes and sets were activated by incubation with PAR2-AP (20 μM). The cells were fixed and probed for TF using an FITC-conjugated anti-TF antibody (HTF1-FITC). The patterns were compared to MAGI1 probed with a rabbit anti-MAGI1 antibody (H-70) and developed a goat anti-rabbit IgG-AlexaFluor 594 antibody. The cells were also stained with DAPI (2 μg/ml) and examined by fluorescence microscopy. Co-localisation coefficient values were then determiend using the ImageJ program. The values were calculated as the average from 10 captured images, and the data show the values for 5 separate experiments

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Incubation, Staining, Fluorescence, Microscopy

Identification of the PDZ domain within MAGI1 responsible for binding TF. MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes and separately transfected with constructs to express PDZ1-5 of from MAGI1. The cells permitted to express the proteins for 24 h and then fixed and washed. A The proximity of the expressed PDZ to TF was examined by PLA using a rabbit anti-HA (C2954) antibody and a mouse anti-TF (HTF1) antibody, respectively (RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI) and ( B ) quantified using the ImageJ program. C Cell surface TF-fVIIa activity was measured on the transfected cells expressing PDZ1, PDZ2 or the empty vector, using a modified thrombin-generation assay. An additional sample of cells were transfected to express PDZ1 but were pre-incubated with an inhibitory anti-TF antibody (HTF1; 20 µg/ml) before analysis. The cells (10 5 ) were incubated for 20 min at 37 °C with a mixture of barium sulphate absorbable proteins (2 mg/ml) and 5 mM CaCl 2 in the reaction buffer (total volume 150 µl; Tris-buffered saline pH 7.4), containing 1% (w/v) bovine serum albumin (BSA). Aliquots of reaction (100 µl) were then transferred to 96-well plates containing 100 µl of thrombin substrate CS-01(38) (0.2 µM H–D-Phe-Pip-Arg-pNA) and incubated for a further 40 min at 37 °C. The reactions were stopped by the addition of 2% (v/v) acetic acid (50 µl) and the absorption of the sample measured at 410 nm on a plate reader

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Identification of the PDZ domain within MAGI1 responsible for binding TF. MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes and separately transfected with constructs to express PDZ1-5 of from MAGI1. The cells permitted to express the proteins for 24 h and then fixed and washed. A The proximity of the expressed PDZ to TF was examined by PLA using a rabbit anti-HA (C2954) antibody and a mouse anti-TF (HTF1) antibody, respectively (RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI) and ( B ) quantified using the ImageJ program. C Cell surface TF-fVIIa activity was measured on the transfected cells expressing PDZ1, PDZ2 or the empty vector, using a modified thrombin-generation assay. An additional sample of cells were transfected to express PDZ1 but were pre-incubated with an inhibitory anti-TF antibody (HTF1; 20 µg/ml) before analysis. The cells (10 5 ) were incubated for 20 min at 37 °C with a mixture of barium sulphate absorbable proteins (2 mg/ml) and 5 mM CaCl 2 in the reaction buffer (total volume 150 µl; Tris-buffered saline pH 7.4), containing 1% (w/v) bovine serum albumin (BSA). Aliquots of reaction (100 µl) were then transferred to 96-well plates containing 100 µl of thrombin substrate CS-01(38) (0.2 µM H–D-Phe-Pip-Arg-pNA) and incubated for a further 40 min at 37 °C. The reactions were stopped by the addition of 2% (v/v) acetic acid (50 µl) and the absorption of the sample measured at 410 nm on a plate reader

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Binding Assay, Transfection, Construct, Activity Assay, Expressing, Plasmid Preparation, Modification, Incubation, Saline

Examination of the role of PDZ1 in interaction with TF. MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes and separately transfected with constructs to express the N-terminal domain of MAGI1 including and excluding PDZ1. The cells were permitted to express the proteins for 24 h and then fixed and washed. A The proximity of the expressed N-terminal peptides to TF was examined by PLA using a rabbit anti-HA (C2954) antibody and a mouse anti-TF (HTF1) antibody, respectively (RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI) and ( B ) quantified using the ImageJ program. C TF was immunoprecipitated from the lysates of the cells expressing the two N-terminal peptides (including and excluding PDZ1) using a mouse anti-HA antibody (C2954). The samples were then examined by western blot and probed using rabbit anti-TF antibody (HTF1). D The relative amounts of TF were then analysed in the samples. E Cell surface TF-fVIIa activity was measured on the transfected cells expressing the N-terminal of MAGI1 with and without the PDZ1 region, or the empty vector, using a modified thrombin-generation assay. The cells (10 5 ) were incubated for 20 min at 37 °C with a mixture of barium sulphate absorbable proteins (2 mg/ml) and 5 mM CaCl 2 in the reaction buffer (total volume 150 µl; Tris-buffered saline (TBS) pH 7.4), containing 1% (w/v) bovine serum albumin (BSA). Aliquots of reaction (100 µl) were then transferred to 96-well plates containing 100 µl of thrombin substrate CS-01(38) (0.2 µM H–D-Phe-Pip-Arg-pNA) and incubated for a further 40 min at 37 °C. The reactions were stopped by the addition of 2% (v/v) acetic acid (50 µl) and the absorption of the sample measured at 410 nm

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Examination of the role of PDZ1 in interaction with TF. MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes and separately transfected with constructs to express the N-terminal domain of MAGI1 including and excluding PDZ1. The cells were permitted to express the proteins for 24 h and then fixed and washed. A The proximity of the expressed N-terminal peptides to TF was examined by PLA using a rabbit anti-HA (C2954) antibody and a mouse anti-TF (HTF1) antibody, respectively (RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI) and ( B ) quantified using the ImageJ program. C TF was immunoprecipitated from the lysates of the cells expressing the two N-terminal peptides (including and excluding PDZ1) using a mouse anti-HA antibody (C2954). The samples were then examined by western blot and probed using rabbit anti-TF antibody (HTF1). D The relative amounts of TF were then analysed in the samples. E Cell surface TF-fVIIa activity was measured on the transfected cells expressing the N-terminal of MAGI1 with and without the PDZ1 region, or the empty vector, using a modified thrombin-generation assay. The cells (10 5 ) were incubated for 20 min at 37 °C with a mixture of barium sulphate absorbable proteins (2 mg/ml) and 5 mM CaCl 2 in the reaction buffer (total volume 150 µl; Tris-buffered saline (TBS) pH 7.4), containing 1% (w/v) bovine serum albumin (BSA). Aliquots of reaction (100 µl) were then transferred to 96-well plates containing 100 µl of thrombin substrate CS-01(38) (0.2 µM H–D-Phe-Pip-Arg-pNA) and incubated for a further 40 min at 37 °C. The reactions were stopped by the addition of 2% (v/v) acetic acid (50 µl) and the absorption of the sample measured at 410 nm

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Transfection, Construct, Immunoprecipitation, Expressing, Western Blot, Activity Assay, Plasmid Preparation, Modification, Incubation, Saline

Outcome of expression of the MAGI1 N-terminal peptides on cell proliferation signalling. MDA-MB-231 cells (10 5 ) were transfected to express the N-terminal of MAGI1 with and without the PDZ1 domain. Selected sets of cells were also pre-incubated with SAM11 antibody (20 µg/ml) as shown in the figures. The cells were lysed and proteins separated by denaturing 12% (w/v) polyacrylamide electrophoresis, transferred onto nitrocellulose membranes and blocked with TBST. A The membranes were probed with a goat anti-human Akt1/2 (N-19) polyclonal antibody and a rabbit polyclonal anti-human Akt1 (phospho-S473). The membranes were then washed and probed with a donkey anti-goat, or goat anti-rabbit alkaline phosphatase-conjugated antibody, diluted 1:4000 (v/v). Bands were visualised using the Western Blue stabilised alkaline phosphatase-substrate, recorded using ImageJ program and ( B ) the ratios calculated. C Separate sets of the western blot membrane were probed using an anti-phosphoT202/185-phosphoY204/187-ERK1/2 antibody or alternatively, total ERK1/2 was detected using an anti-ERK1/2 antibody diluted 1:3000 (v/v) in TBST. The membranes were also probed using a rabbit anti-GAPDH polyclonal antibody (V-18) diluted 1:5000 (v/v) in TBST. The membranes were then incubated with a goat anti-rabbit alkaline or a donkey anti-goat phosphatase-conjugated antibody diluted 1:5000 (v/v) in TBST and visualised as above, recorded using ImageJ program and ( D ) the ratios calculated. MDA-MB-231 cells (10 5 ) were transfected to express the N-terminal of MAGI1 with and without the PDZ1 domain. Sets of cells were pre-incubated with a monoclonal antibody (SAM11; 20 µg/ml) to block PAR2 activation, or a mouse monoclonal antibody capable of inhibiting the protease activity of TF-fVIIa complex (HTF1; 20 µg/ml). E Total RNA was isolated from one set of the cells, and samples (100 ng) were amplified using the primers 5’- CCG TCC ATG CGG AAG ATC -3’ (forward) and 5’- ATG GCC AGC GGG AAG AC -3’ (reverse). The reaction was carried out at an annealing temperature of 60 °C using the GoTaq® 1-Step RT-qPCR for 40 cycles. Following amplification, the relative amounts of target mRNA were determined using the 2 −ΔΔCT method. F Cell numbers were determined in the second sets of cells using the crystal violet method

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Outcome of expression of the MAGI1 N-terminal peptides on cell proliferation signalling. MDA-MB-231 cells (10 5 ) were transfected to express the N-terminal of MAGI1 with and without the PDZ1 domain. Selected sets of cells were also pre-incubated with SAM11 antibody (20 µg/ml) as shown in the figures. The cells were lysed and proteins separated by denaturing 12% (w/v) polyacrylamide electrophoresis, transferred onto nitrocellulose membranes and blocked with TBST. A The membranes were probed with a goat anti-human Akt1/2 (N-19) polyclonal antibody and a rabbit polyclonal anti-human Akt1 (phospho-S473). The membranes were then washed and probed with a donkey anti-goat, or goat anti-rabbit alkaline phosphatase-conjugated antibody, diluted 1:4000 (v/v). Bands were visualised using the Western Blue stabilised alkaline phosphatase-substrate, recorded using ImageJ program and ( B ) the ratios calculated. C Separate sets of the western blot membrane were probed using an anti-phosphoT202/185-phosphoY204/187-ERK1/2 antibody or alternatively, total ERK1/2 was detected using an anti-ERK1/2 antibody diluted 1:3000 (v/v) in TBST. The membranes were also probed using a rabbit anti-GAPDH polyclonal antibody (V-18) diluted 1:5000 (v/v) in TBST. The membranes were then incubated with a goat anti-rabbit alkaline or a donkey anti-goat phosphatase-conjugated antibody diluted 1:5000 (v/v) in TBST and visualised as above, recorded using ImageJ program and ( D ) the ratios calculated. MDA-MB-231 cells (10 5 ) were transfected to express the N-terminal of MAGI1 with and without the PDZ1 domain. Sets of cells were pre-incubated with a monoclonal antibody (SAM11; 20 µg/ml) to block PAR2 activation, or a mouse monoclonal antibody capable of inhibiting the protease activity of TF-fVIIa complex (HTF1; 20 µg/ml). E Total RNA was isolated from one set of the cells, and samples (100 ng) were amplified using the primers 5’- CCG TCC ATG CGG AAG ATC -3’ (forward) and 5’- ATG GCC AGC GGG AAG AC -3’ (reverse). The reaction was carried out at an annealing temperature of 60 °C using the GoTaq® 1-Step RT-qPCR for 40 cycles. Following amplification, the relative amounts of target mRNA were determined using the 2 −ΔΔCT method. F Cell numbers were determined in the second sets of cells using the crystal violet method

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Expressing, Transfection, Incubation, Electrophoresis, Western Blot, Membrane, Blocking Assay, Activation Assay, Activity Assay, Isolation, Amplification, Quantitative RT-PCR

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Proximity Ligation Assay, Activation Assay, Incubation, Blocking Assay, Fluorescence, Microscopy

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Incubation, Staining, Fluorescence, Microscopy

Examination of influence of TF phosphorylation on the binding of MAGI1 and 3. Biotinylated peptides, corresponding to the last 18 amino acids of the cytoplasmic domain of TF were synthesised in non-phosphorylated, single-phosphorylated and double-phosphorylated forms and used as bait. An additional scrambled peptide (biotin-SWGNVSKLSAPRQGVNKE) was also included alongside. The peptides (5 µM final concentration) in PBS, were bound into a NeutrAvidin-coated 96-well plate and then blocked. Cell lysates from resting and PAR2-activated MDA-MB-231 cells (from 2 × 10 5 cells) were incubated in the plates for 1 h at room temperature. The wells were then washed four times and probed with ( A ) rabbit anti-MAGI1 (H-70) or ( B ) mouse anti-MAGI3 (46) antibodies diluted 1:200 (v/v) in PBST. The samples were detected using goat anti-rabbit and goat anti-mouse alkaline phosphatase-conjugated antibodies diluted 1:200 (v/v) and the colour developed with TMB One Solution (100 µl). Once the colour was developed the reactions were stopped and absorptions recorded. ( n = 3; * = p < 0.05 vs. the respective samples without cell lysate). C The interaction of MAGI1 with the non-phosphorylated TF peptide was examined by pull-down over a period of 30 min following activation of PAR2, as described above

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Examination of influence of TF phosphorylation on the binding of MAGI1 and 3. Biotinylated peptides, corresponding to the last 18 amino acids of the cytoplasmic domain of TF were synthesised in non-phosphorylated, single-phosphorylated and double-phosphorylated forms and used as bait. An additional scrambled peptide (biotin-SWGNVSKLSAPRQGVNKE) was also included alongside. The peptides (5 µM final concentration) in PBS, were bound into a NeutrAvidin-coated 96-well plate and then blocked. Cell lysates from resting and PAR2-activated MDA-MB-231 cells (from 2 × 10 5 cells) were incubated in the plates for 1 h at room temperature. The wells were then washed four times and probed with ( A ) rabbit anti-MAGI1 (H-70) or ( B ) mouse anti-MAGI3 (46) antibodies diluted 1:200 (v/v) in PBST. The samples were detected using goat anti-rabbit and goat anti-mouse alkaline phosphatase-conjugated antibodies diluted 1:200 (v/v) and the colour developed with TMB One Solution (100 µl). Once the colour was developed the reactions were stopped and absorptions recorded. ( n = 3; * = p < 0.05 vs. the respective samples without cell lysate). C The interaction of MAGI1 with the non-phosphorylated TF peptide was examined by pull-down over a period of 30 min following activation of PAR2, as described above

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Phospho-proteomics, Binding Assay, Concentration Assay, Incubation, Activation Assay

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Binding Assay, Transfection, Construct, Activity Assay, Expressing, Plasmid Preparation, Modification, Incubation, Saline

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Transfection, Construct, Immunoprecipitation, Expressing, Western Blot, Activity Assay, Plasmid Preparation, Modification, Incubation, Saline

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Expressing, Transfection, Incubation, Electrophoresis, Western Blot, Membrane, Blocking Assay, Activation Assay, Activity Assay, Isolation, Amplification, Quantitative RT-PCR

Analysis of the interaction of TF and MAGI1-3 and the influence of PAR2 activation. A MDA-MB-231 cells (2 × 10 5 ) were adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 20 min and TF was immunoprecipitated from cell lysates with the anti-TF (HTF-1; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for MAGI1 and TF by western blot using an anti-MAGI1 (H-70) and a rabbit anti-TF antibody (FL-295). B The ratio of the MAGI1 band densities were normalised against those of TF in the same co-immunoprecipitated samples. C In addition, MAGI1 was immunoprecipitated from cell lysates with an anti-MAGI1 (H-70; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for TF and MAGI1 by western blot using an anti-TF antibody (HTF-1) and a mouse anti-MAGI1 antibody (SS-5). D The ratios of the TF band densities were normalised against those of MAGI1 in the same co-immunoprecipitated samples

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Analysis of the interaction of TF and MAGI1-3 and the influence of PAR2 activation. A MDA-MB-231 cells (2 × 10 5 ) were adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 20 min and TF was immunoprecipitated from cell lysates with the anti-TF (HTF-1; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for MAGI1 and TF by western blot using an anti-MAGI1 (H-70) and a rabbit anti-TF antibody (FL-295). B The ratio of the MAGI1 band densities were normalised against those of TF in the same co-immunoprecipitated samples. C In addition, MAGI1 was immunoprecipitated from cell lysates with an anti-MAGI1 (H-70; 4 µg) antibody using protein A-magnetic beads. The samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and examined for TF and MAGI1 by western blot using an anti-TF antibody (HTF-1) and a mouse anti-MAGI1 antibody (SS-5). D The ratios of the TF band densities were normalised against those of MAGI1 in the same co-immunoprecipitated samples

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Activation Assay, Incubation, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot

Schematic representation of the regulation of TF activity by interaction with MAGI1. A The interaction of the cytoplasmic domain of TF with the PDZ1 domain of MAGI1 restrains TF. Initiation of cell signalling following the activation of PAR2 on the cell surface may result in ( B ) the induction of capsase-1 leading to the degradation on MAGI1 into non-functional fragments. C Concurrent phosphorylation of TF by PKC at Ser253 prevents further binding with other MAGI1 proteins on the cells surface. These alterations collectively may promote the processing of TF permitting interaction with fVIIa as an active complex

Journal: Thrombosis Journal

Article Title: Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

doi: 10.1186/s12959-023-00580-6

Figure Lengend Snippet: Schematic representation of the regulation of TF activity by interaction with MAGI1. A The interaction of the cytoplasmic domain of TF with the PDZ1 domain of MAGI1 restrains TF. Initiation of cell signalling following the activation of PAR2 on the cell surface may result in ( B ) the induction of capsase-1 leading to the degradation on MAGI1 into non-functional fragments. C Concurrent phosphorylation of TF by PKC at Ser253 prevents further binding with other MAGI1 proteins on the cells surface. These alterations collectively may promote the processing of TF permitting interaction with fVIIa as an active complex

Article Snippet: When immunoprecipitating with anti-MAGI1, the membranes were probed with an anti-TF antibody (HTF1) and a mouse anti-MAGI1 antibody (SS-5; Santa Cruz).

Techniques: Activity Assay, Activation Assay, Functional Assay, Phospho-proteomics, Binding Assay

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