profluor® src-family kinase assay  (Promega)

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, kinase activity and induction of cell apoptosis. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of pCMV6-Ac-tGFP, pCMV6-Ac-TF Ala253 -tGFP, pCMV6-Ac-TF-tGFP plasmids, along with an untransfected set of cells. 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) for 90 min. a The cells were then lysed and phosphorylated and total Src1 examined by western blot, as described before. b The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 4; * = p < 0.05 vs. the untreated samples). c HCAEC (2 × 10 5 /well) were transfected as above and incubated for 48 h to permit protein expression. Cells were then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min and activated with PAR2-AP (20 μM) for a further 90 min. The cells were then lysed in PhosphoSafe Extraction Reagent and the Src activity measured using the ProFluor ® Src-family kinase assay. The fluorescence intensity of the released rhodamine was measured at Em 530 nm (Ex 485 nm), using the absorption of the included AMC at Em 460 nm (Ex 355 nm), as reference for the initial substrate concentration. (n = 5; * = p < 0.05 vs. the untreated samples). d HCAEC (2 × 10 5 /well) were transfected as above and incubated for 48 h to permit protein expression. Cells were then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min and activated with PAR2-AP (20 μM) for a further 90 min. The rate of cellular apoptosis was then quantified at 20 h, using the TiterTACS™ Colorimetric Apoptosis Detection Kit. (n = 4; * = p < 0.05 vs. the untreated samples)

Article Snippet: The ProFluor ® Src-family kinase assay (Promega Corporation, Southampton, UK) was used to quantify the Src1 enzymatic activity within the cells.

Techniques: Phospho-proteomics, Activity Assay, Transfection, Incubation, Expressing, Recombinant, Western Blot, Extraction, Kinase Assay, Fluorescence, Concentration Assay

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: Examination of the involvement of FAK in TF-mediated induction of cell apoptosis. Sets of HCAEC (2 × 10 5 /well) were transfected with pCMV6-Ac-tGFP, pCMV6-Ac-TF Ala253 -tGFP, pCMV6-Ac-TF-tGFP plasmids, along with an untransfected set of cells and incubated for 48 h to permit protein expression. Cells were then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min together with FAK inhibitor-14 (100 µM) and compared to vehicle control. The cells were then activated with PAR2-AP (20 μM) for a further 90 min. a The cells were then lysed and phosphorylated and total Src1 examined by western blot, as described before. b The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 4; * = p < 0.05 vs. the untreated samples). c Phosphorylation of FAK was also analysed by western blot using specific antibodies to Tyr397-phosphorylated and total FAK. d The ratios of phosphorylated to total FAK were calculated using the ImageJ program. (n = 4; * = p < 0.05 vs. the untreated samples). e Sets of cells were treated as above and then lysed in PhosphoSafe Extraction Reagent and the Src kinase activity measured using the ProFluor ® Src-family kinase assay. (n = 4). f In addition, the rate of cellular apoptosis was then quantified at 20 h, using the TiterTACS™ Colorimetric Apoptosis Detection Kit. (n = 4). g Sets of HCAEC were transfected and adapted to serum-free medium as above. One set of cells were then activated with PAR2-AP (20 µM) and the level of FAK phosphorylation by western blot. h The ratios of phosphorylated to total FAK were calculated using the ImageJ program. (n = 4)

Article Snippet: The ProFluor ® Src-family kinase assay (Promega Corporation, Southampton, UK) was used to quantify the Src1 enzymatic activity within the cells.

Techniques: Transfection, Incubation, Expressing, Control, Western Blot, Phospho-proteomics, Extraction, Activity Assay, Kinase Assay

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: Examination of the involvement of β1-integrin in TF-mediated induction of cell apoptosis. Sets of HCAEC (2 × 10 5 /well) were transfected with pCMV6-Ac-tGFP, pCMV6-Ac-TF Ala253 -tGFP, pCMV6-Ac-TF-tGFP plasmids, along with an untransfected set of cells and incubated for 48 h to permit protein expression. Cells were then adapted to low-serum medium MV containing 2% (v/v) FCS for 60 min together with a blocking anti-β1-integrin antibody (AIIB2; 20 µg/ml) and then were activated with PAR2-AP (20 μM) for a further 90 min. a The cells were then lysed and phosphorylated and total Src1 examined by western blot, as described before. b The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 4; * = p < 0.05 vs. untreated samples; # = p < 0.05 vs. respective control IgG). c Sets of cells were treated as above and then lysed in PhosphoSafe Extraction Reagent and the Src kinase activity measured using the ProFluor ® Src-family kinase assay. (n = 4; * = p < 0.05 vs. the untreated samples; # = p < 0.05 vs. respective control IgG). d Sets of cells were treated as above and the rate of cellular apoptosis was quantified at 20 h, using the TiterTACS™ Colorimetric Apoptosis Detection Kit. (n = 4; * = p < 0.05 vs. the untreated samples; # = p < 0.05 vs. respective control IgG)

Article Snippet: The ProFluor ® Src-family kinase assay (Promega Corporation, Southampton, UK) was used to quantify the Src1 enzymatic activity within the cells.

Techniques: Transfection, Incubation, Expressing, Blocking Assay, Western Blot, Control, Extraction, Activity Assay, Kinase Assay

Journal: Developmental Cell

Article Title: The Ciliary Machinery Is Repurposed for T Cell Immune Synapse Trafficking of LCK

doi: 10.1016/j.devcel.2018.08.012

Figure Lengend Snippet:

Article Snippet: Kinase activity of wild-type and mutant versions of LCK was measured using the ProFluor® Src-Family Kinase Assay kit (Promega), as per the manufacturer’s instructions and measured using a Tecan Safire 2 multi-mode plate reader (ThermoFisher).

Techniques: Virus, Recombinant, Kinase Assay, Software

Journal: Developmental Cell

Article Title: The Ciliary Machinery Is Repurposed for T Cell Immune Synapse Trafficking of LCK

doi: 10.1016/j.devcel.2018.08.012

Figure Lengend Snippet:

Article Snippet: ProFluor® Src-Family Kinase Assay , Promega , V1270.

Techniques: Virus, Recombinant, Kinase Assay, Software