rabbit polyclonal anti cpla2α  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification

doi: 10.3390/ijms21176398

Figure Lengend Snippet: Quantification of cPLA2α in cultured AVICs. ( a ) Western blotting quantification of cPLA2α in 3- to 9-day-long control and metastatic calcification-like cultures. Protein levels are calculated as cPLA2α/β-actin, with the control being set to 1. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( b ) Western blotting quantification of cPLA2α in 9- to 28-day-long control and severe dystrophic calcification-like cultures. Protein levels are calculated as cPLA2α/β-actin, with the control being set to 1. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( c ) Representative Western blotting obtained from lysates of AVICs cultured as in ( a ) and ( b ). ( d ) Quantification of cPLA2α mRNA using qPCR in 9- to 28-day-long control and severe dystrophic calcification-like cultures. Data are expressed as 2^-ddCT. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). Abbreviations: AVIC, aortic valve interstitial cell; Pi, phosphate; LPS, lipopolysaccharide; CM, conditioned medium.

Article Snippet: On expiry of each incubation time, AVICs were fixed with 3% PBS-buffered paraformaldehyde for 10 min and then treated with (i) 0.1% PBS-diluted Triton X-100 for 10 min, (ii) 3% PBS-diluted hydrogen peroxide for 5 min, (iii) 3% PBS-diluted normal serum for 40 min, (iv) 1:100 PBS-diluted rabbit anti-cPLA2α polyclonal antibody (Novus Biologicals; Cat. #: NBP2-19809; recombinant protein encompassing a sequence within the center region of human PLA2G4A) for 90 min at r.t., (v) 1:600 PBS-diluted peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch; Cat. #: 711-036-152) for 30 min, and (vi) DAB chromogen (BioGenex, Fremont, CA, USA) prepared according to the manufacturer’s instructions for 6 min. As endogenous control, primary antibody was replaced with normal serum.

Techniques: Cell Culture, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification

doi: 10.3390/ijms21176398

Figure Lengend Snippet: Percentages of cPLA2α-immunopositive AVICs. ( a ) Percentages of immunopositive cells in 3- to 9-day-long control and metastatic calcification-like cultures. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( b ) Percentages of immunopositive cells in 9- to 28-day-long control and severe dystrophic calcification-like cultures. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). Abbreviations: AVICs, aortic valve interstitial cells; Pi, phosphate; LPS, lipopolysaccharide; CM, conditioned medium. ( a1 – a3 ) Representative light microscopy micrographs of 3- to 9-day-long metastatic calcification-like cultures immunoreacted for cPLA2α. ( a4 – a6 ) Representative light microscopy micrographs of 3- to 9-day-long control cultures immunoreacted for cPLA2α. ( b1 – b4 ) Representative light microscopy micrographs of 9- to 28-day-long severe dystrophic calcification-like cultures immunoreacted for cPLA2α. ( b5 – b8 ) Representative light microscopy micrographs of 9- to 28-day-long control cultures immunoreacted for cPLA2α; Bar: 1 mm.

Article Snippet: On expiry of each incubation time, AVICs were fixed with 3% PBS-buffered paraformaldehyde for 10 min and then treated with (i) 0.1% PBS-diluted Triton X-100 for 10 min, (ii) 3% PBS-diluted hydrogen peroxide for 5 min, (iii) 3% PBS-diluted normal serum for 40 min, (iv) 1:100 PBS-diluted rabbit anti-cPLA2α polyclonal antibody (Novus Biologicals; Cat. #: NBP2-19809; recombinant protein encompassing a sequence within the center region of human PLA2G4A) for 90 min at r.t., (v) 1:600 PBS-diluted peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch; Cat. #: 711-036-152) for 30 min, and (vi) DAB chromogen (BioGenex, Fremont, CA, USA) prepared according to the manufacturer’s instructions for 6 min. As endogenous control, primary antibody was replaced with normal serum.

Techniques: Light Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification

doi: 10.3390/ijms21176398

Figure Lengend Snippet: Effects of cPLA2α inhibitor dexamethasone on pro-calcific AVIC cultures. ( a ) Western blotting quantification of cPLA2α in 6-day-long control and metastatic calcification-like cultures with or without dexamethasone supplementation. Protein levels are calculated as cPLA2α/β-actin, with the control being set to 1. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( b ) Western blotting quantification of cPLA2α in 21-day-long control and severe dystrophic calcification-like cultures with or without dexamethasone supplementation. Protein levels are calculated as cPLA2α/β-actin, with the control being set to 1. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( a1 ) Representative Western blotting obtained from lysates of AVICs cultured as in a. ( b1 ) Representative Western blotting obtained from lysates of AVICs cultured as in ( b ). ( c ) Quantification of cPLA2α mRNA using qPCR in 21-day-long control and severe dystrophic calcification-like cultures with or without dexamethasone supplementation. Data are expressed as 2^-ddCT. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). Abbreviations: AVIC, aortic valve interstitial cell; Pi, phosphate; LPS, lipopolysaccharide; CM, conditioned medium; Dex, dexamethasone.

Article Snippet: On expiry of each incubation time, AVICs were fixed with 3% PBS-buffered paraformaldehyde for 10 min and then treated with (i) 0.1% PBS-diluted Triton X-100 for 10 min, (ii) 3% PBS-diluted hydrogen peroxide for 5 min, (iii) 3% PBS-diluted normal serum for 40 min, (iv) 1:100 PBS-diluted rabbit anti-cPLA2α polyclonal antibody (Novus Biologicals; Cat. #: NBP2-19809; recombinant protein encompassing a sequence within the center region of human PLA2G4A) for 90 min at r.t., (v) 1:600 PBS-diluted peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch; Cat. #: 711-036-152) for 30 min, and (vi) DAB chromogen (BioGenex, Fremont, CA, USA) prepared according to the manufacturer’s instructions for 6 min. As endogenous control, primary antibody was replaced with normal serum.

Techniques: Western Blot, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification

doi: 10.3390/ijms21176398

Figure Lengend Snippet: Quantification of cPLA2α in cultured AVICs. ( a ) Western blotting quantification of cPLA2α in 3- to 9-day-long control and metastatic calcification-like cultures. Protein levels are calculated as cPLA2α/β-actin, with the control being set to 1. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( b ) Western blotting quantification of cPLA2α in 9- to 28-day-long control and severe dystrophic calcification-like cultures. Protein levels are calculated as cPLA2α/β-actin, with the control being set to 1. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( c ) Representative Western blotting obtained from lysates of AVICs cultured as in ( a ) and ( b ). ( d ) Quantification of cPLA2α mRNA using qPCR in 9- to 28-day-long control and severe dystrophic calcification-like cultures. Data are expressed as 2^-ddCT. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). Abbreviations: AVIC, aortic valve interstitial cell; Pi, phosphate; LPS, lipopolysaccharide; CM, conditioned medium.

Article Snippet: On expiry of each incubation time, AVICs were fixed with 3% PBS-buffered paraformaldehyde for 10 min and then treated with (i) 0.1% PBS-diluted Triton X-100 for 10 min, (ii) 3% PBS-diluted hydrogen peroxide for 5 min, (iii) 3% PBS-diluted normal serum for 40 min, (iv) 1:100 PBS-diluted rabbit anti-cPLA2α polyclonal antibody (Novus Biologicals; Cat. #: NBP2-19809; recombinant protein encompassing a sequence within the center region of human PLA2G4A) for 90 min at r.t., (v) 1:600 PBS-diluted peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch; Cat. #: 711-036-152) for 30 min, and (vi) DAB chromogen (BioGenex, Fremont, CA, USA) prepared according to the manufacturer’s instructions for 6 min. As endogenous control, primary antibody was replaced with normal serum.

Techniques: Cell Culture, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification

doi: 10.3390/ijms21176398

Figure Lengend Snippet: Percentages of cPLA2α-immunopositive AVICs. ( a ) Percentages of immunopositive cells in 3- to 9-day-long control and metastatic calcification-like cultures. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( b ) Percentages of immunopositive cells in 9- to 28-day-long control and severe dystrophic calcification-like cultures. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). Abbreviations: AVICs, aortic valve interstitial cells; Pi, phosphate; LPS, lipopolysaccharide; CM, conditioned medium. ( a1 – a3 ) Representative light microscopy micrographs of 3- to 9-day-long metastatic calcification-like cultures immunoreacted for cPLA2α. ( a4 – a6 ) Representative light microscopy micrographs of 3- to 9-day-long control cultures immunoreacted for cPLA2α. ( b1 – b4 ) Representative light microscopy micrographs of 9- to 28-day-long severe dystrophic calcification-like cultures immunoreacted for cPLA2α. ( b5 – b8 ) Representative light microscopy micrographs of 9- to 28-day-long control cultures immunoreacted for cPLA2α; Bar: 1 mm.

Article Snippet: On expiry of each incubation time, AVICs were fixed with 3% PBS-buffered paraformaldehyde for 10 min and then treated with (i) 0.1% PBS-diluted Triton X-100 for 10 min, (ii) 3% PBS-diluted hydrogen peroxide for 5 min, (iii) 3% PBS-diluted normal serum for 40 min, (iv) 1:100 PBS-diluted rabbit anti-cPLA2α polyclonal antibody (Novus Biologicals; Cat. #: NBP2-19809; recombinant protein encompassing a sequence within the center region of human PLA2G4A) for 90 min at r.t., (v) 1:600 PBS-diluted peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch; Cat. #: 711-036-152) for 30 min, and (vi) DAB chromogen (BioGenex, Fremont, CA, USA) prepared according to the manufacturer’s instructions for 6 min. As endogenous control, primary antibody was replaced with normal serum.

Techniques: Control, Light Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification

doi: 10.3390/ijms21176398

Figure Lengend Snippet: Effects of cPLA2α inhibitor dexamethasone on pro-calcific AVIC cultures. ( a ) Western blotting quantification of cPLA2α in 6-day-long control and metastatic calcification-like cultures with or without dexamethasone supplementation. Protein levels are calculated as cPLA2α/β-actin, with the control being set to 1. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( b ) Western blotting quantification of cPLA2α in 21-day-long control and severe dystrophic calcification-like cultures with or without dexamethasone supplementation. Protein levels are calculated as cPLA2α/β-actin, with the control being set to 1. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). ( a1 ) Representative Western blotting obtained from lysates of AVICs cultured as in a. ( b1 ) Representative Western blotting obtained from lysates of AVICs cultured as in ( b ). ( c ) Quantification of cPLA2α mRNA using qPCR in 21-day-long control and severe dystrophic calcification-like cultures with or without dexamethasone supplementation. Data are expressed as 2^-ddCT. Data are shown as mean ± SE. Statistically significant values are indicated with asterisks ( p < 0.05). Abbreviations: AVIC, aortic valve interstitial cell; Pi, phosphate; LPS, lipopolysaccharide; CM, conditioned medium; Dex, dexamethasone.

Article Snippet: On expiry of each incubation time, AVICs were fixed with 3% PBS-buffered paraformaldehyde for 10 min and then treated with (i) 0.1% PBS-diluted Triton X-100 for 10 min, (ii) 3% PBS-diluted hydrogen peroxide for 5 min, (iii) 3% PBS-diluted normal serum for 40 min, (iv) 1:100 PBS-diluted rabbit anti-cPLA2α polyclonal antibody (Novus Biologicals; Cat. #: NBP2-19809; recombinant protein encompassing a sequence within the center region of human PLA2G4A) for 90 min at r.t., (v) 1:600 PBS-diluted peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch; Cat. #: 711-036-152) for 30 min, and (vi) DAB chromogen (BioGenex, Fremont, CA, USA) prepared according to the manufacturer’s instructions for 6 min. As endogenous control, primary antibody was replaced with normal serum.

Techniques: Western Blot, Control, Cell Culture