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Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted <t>1:3000</t> (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in <t>TBST.</t> The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
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1) Product Images from "Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling"

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

Journal: Apoptosis

doi: 10.1007/s10495-019-01576-2

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

2) Product Images from "Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling"

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

Journal: Apoptosis

doi: 10.1007/s10495-019-01576-2

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

3) Product Images from "Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling"

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

Journal: Apoptosis

doi: 10.1007/s10495-019-01576-2

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

4) Product Images from "Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling"

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

Journal: Apoptosis

doi: 10.1007/s10495-019-01576-2

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

5) Product Images from "Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling"

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

Journal: Apoptosis

doi: 10.1007/s10495-019-01576-2

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

6) Product Images from "Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling"

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

Journal: Apoptosis

doi: 10.1007/s10495-019-01576-2

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

7) Product Images from "Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling"

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

Journal: Apoptosis

doi: 10.1007/s10495-019-01576-2

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

8) Product Images from "Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling"

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

Journal: Apoptosis

doi: 10.1007/s10495-019-01576-2

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p
Figure Legend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

Techniques Used: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

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    Cell Signaling Technology Inc tbst
    Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted <t>1:3000</t> (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in <t>TBST.</t> The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p
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    Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

    Journal: Apoptosis

    Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

    doi: 10.1007/s10495-019-01576-2

    Figure Lengend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p

    Article Snippet: Total and phosphorylated FAK were detected using a polyclonal rabbit anti-human FAK diluted 1:3000 (v/v) in TBST, and a monoclonal rabbit anti-human phospho-Tyr397-FAK (D20B1) diluted 1:3000 (v/v) in TBST, respectively (Cell Signalling Technologies).

    Techniques: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

    Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

    Journal: Apoptosis

    Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

    doi: 10.1007/s10495-019-01576-2

    Figure Lengend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p

    Article Snippet: Total and phosphorylated FAK were detected using a polyclonal rabbit anti-human FAK diluted 1:3000 (v/v) in TBST, and a monoclonal rabbit anti-human phospho-Tyr397-FAK (D20B1) diluted 1:3000 (v/v) in TBST, respectively (Cell Signalling Technologies).

    Techniques: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page