recombinant human parp-1 enzyme (high specific activity)  (Trevigen)

Journal: The American Journal of Pathology

Article Title: Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

doi: 10.1016/j.ajpath.2012.09.004

Figure Lengend Snippet: Effect of PARP-1 inhibitor on myofibroblast differentiation. A: Rat lung fibroblasts were treated with TGF-β at 4 ng/mL or buffer only (None) and the PARP-1 inhibitor 3-AB at concentrations from 0 to 12 mmol/L for 48 hours and then were analyzed for α-SMA mRNA by qPCR. 18S rRNA was used as the reference, and the level in untreated control cells (without 3-AB) was used as the calibrator. B: Representative phase-contrast images of rat lung fibroblast after treatment for 72 hours with buffer only (top panel) or 10 mmol/L 3-AB (bottom panel) show no appreciable cell toxicity. Data are expressed as means ± SEM. *P < 0.05 versus control. n = 3. Original magnification, ×100.

Article Snippet: Selected samples were pretreated with high-specific-activity PARP-1 enzyme (4668-100-01; Trevigen, Gaithersburg, MD) in 1× PARP buffer (4671-096-02; Trevigen) containing the NAD + substrate of PARP-1 before addition of the 32 P-labeled probe (20,000 to 30,000 counts/minute).

Techniques:

Journal: The American Journal of Pathology

Article Title: Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

doi: 10.1016/j.ajpath.2012.09.004

Figure Lengend Snippet: PARP-1 regulation of α-SMA expression. A: PARP-1 cDNA plasmid (pCMV-sport6-PARP-1), PARP-1 shRNA expression construct (pGIPZ-shPARP-1), or their corresponding empty vector control (Vector only) were transfected into rat lung fibroblasts. The cells were treated with buffer only (None) or with 4 ng/mL TGF-β and then were analyzed for α-SMA mRNA by qPCR analysis 12 hours after transfection. 18S rRNA was used as reference, and the level in untreated cells transfected with vector only was used as the calibrator. B–E: Cells were analyzed for protein by Western blot analysis for α-SMA, PARP-1, and GAPDH 72 hours after transfection. Protein extracts from cells transfected with PARP-1 cDNA or the corresponding vector (B and C) or transfected with PARP-1 shRNA or the corresponding vector (D and E) were analyzed by Western blot analysis for α-SMA and PARP-1 expression. The membrane was reblotted with anti-GAPDH antibody for loading control. Relative density of the bands from three independent experiments was quantified. Data are expressed as means ± SEM (A, C, and E), normalized to the density of corresponding GAPDH bands (C and E). n = 3. *P < 0.05 versus control.

Article Snippet: Selected samples were pretreated with high-specific-activity PARP-1 enzyme (4668-100-01; Trevigen, Gaithersburg, MD) in 1× PARP buffer (4671-096-02; Trevigen) containing the NAD + substrate of PARP-1 before addition of the 32 P-labeled probe (20,000 to 30,000 counts/minute).

Techniques: Expressing, Plasmid Preparation, shRNA, Construct, Transfection, Western Blot, Membrane

Journal: The American Journal of Pathology

Article Title: Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

doi: 10.1016/j.ajpath.2012.09.004

Figure Lengend Snippet: PARP-1 regulation of α-SMA gene transcription. PARP-1 cDNA plasmid, PARP-1 shRNA expression construct, or empty vector (Vector only) were transfected together with the WT rat α-SMA promoter-luciferase fusion plasmid, pGL3-α-SMApro-Luc, into rat lung fibroblasts. Cells were treated with buffer only (None) or TGF-β and analyzed for α-SMA promoter activity. The effects of PARP-1 cDNA (A) or PARP-1 shRNA (B) on α-SMA promoter activity were determined in terms of luciferase activities. Data are expressed as means ± SEM, normalized to the respective Renilla luciferase control activity (to correct for transfection efficiency). n = 3. *P < 0.05 versus control.

Article Snippet: Selected samples were pretreated with high-specific-activity PARP-1 enzyme (4668-100-01; Trevigen, Gaithersburg, MD) in 1× PARP buffer (4671-096-02; Trevigen) containing the NAD + substrate of PARP-1 before addition of the 32 P-labeled probe (20,000 to 30,000 counts/minute).

Techniques: Plasmid Preparation, shRNA, Expressing, Construct, Transfection, Luciferase, Activity Assay

Journal: The American Journal of Pathology

Article Title: Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

doi: 10.1016/j.ajpath.2012.09.004

Figure Lengend Snippet: Binding of Smad3 to Smad3 binding element in the rat α-SMA gene. Double-stranded oligo DNA primers containing the Smad3 binding element in the rat α-SMA gene were synthesized and labeled with 32P for use in an electrophoretic mobility shift assay. Nuclear extracts (2 μg) from PARP-1–deficient (KO) or WT mouse lung fibroblasts (A) or 50 ng of recombinant Smad3 (B) were incubated with the 32P-labeled probe and then analyzed by gel electrophoresis. Nuclear extracts from PARP-1 KO cells (A) or purified Smad3 (B) were pretreated with PARP-1 enzyme without or with the PARP-1 cofactor (NAD+), or with buffer only, before addition of the probe. Samples with the PARP-1 treatment only (without nuclear extract or Smad3) were also incubated with the 32P-labeled probe, as controls. To document binding specificity, excess cold probe (100×) was added to the reaction mixture. The protein-DNA complexes formed by nuclear extract (A) or Smad3 (B) with the labeled oligo DNA were indicated by arrows.

Article Snippet: Selected samples were pretreated with high-specific-activity PARP-1 enzyme (4668-100-01; Trevigen, Gaithersburg, MD) in 1× PARP buffer (4671-096-02; Trevigen) containing the NAD + substrate of PARP-1 before addition of the 32 P-labeled probe (20,000 to 30,000 counts/minute).

Techniques: Binding Assay, Synthesized, Labeling, Electrophoretic Mobility Shift Assay, Recombinant, Incubation, Nucleic Acid Electrophoresis, Purification

Journal: The American Journal of Pathology

Article Title: Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

doi: 10.1016/j.ajpath.2012.09.004

Figure Lengend Snippet: Methylation at individual CpG sites. Genomic DNA isolated from either WT (parp1+/+) or PARP-1-deficient (parp1−/−) murine lung fibroblasts was analyzed for DNA methylation by DNA pyrosequencing with primers specific for the mouse α-SMA gene. The CpG sites are numbered relative to the transcription start site in the mouse α-SMA gene. Data are expressed as means ± SEM. n = 3. *P < 0.05 versus WT control.

Article Snippet: Selected samples were pretreated with high-specific-activity PARP-1 enzyme (4668-100-01; Trevigen, Gaithersburg, MD) in 1× PARP buffer (4671-096-02; Trevigen) containing the NAD + substrate of PARP-1 before addition of the 32 P-labeled probe (20,000 to 30,000 counts/minute).

Techniques: Methylation, Isolation, DNA Methylation Assay

Journal: The American Journal of Pathology

Article Title: Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

doi: 10.1016/j.ajpath.2012.09.004

Figure Lengend Snippet: In vivo effects of PARP-1 deficiency. PARP-1 KO mice and their WT controls were treated with saline (sal) or bleomycin (blm) to induce lung injury and fibrosis. The lungs were then harvested for analysis of fibrosis (total hydroxyproline content) (A) and for myofibroblast differentiation (α-SMA mRNA and protein) and matrix synthesis (type I collagen mRNA and protein) (B–D). A: Collagen deposition was evaluated by hydroxyproline analysis 21 days after bleomycin treatment. B: Lung α-SMA and α1(I) procollagen mRNAs were detected by qPCR using the 2−ΔΔCT method. 18S rRNA was used as reference, and the level in saline-treated WT mice was used as the calibrator. C: Lung α-SMA and α1(I) procollagen proteins were analyzed by Western blotting. Each lane represents a sample from a single animal. GAPDH was used as a loading control, after stripping of the membrane. D: Relative density of the bands was quantified from three independent experiments, normalized to the density of corresponding GAPDH bands. Data are expressed as means ± SEM. n = 5 (A); n = 3 (B–D). *P < 0.05.

Article Snippet: Selected samples were pretreated with high-specific-activity PARP-1 enzyme (4668-100-01; Trevigen, Gaithersburg, MD) in 1× PARP buffer (4671-096-02; Trevigen) containing the NAD + substrate of PARP-1 before addition of the 32 P-labeled probe (20,000 to 30,000 counts/minute).

Techniques: In Vivo, Saline, Western Blot, Stripping Membranes, Membrane

Journal: The American Journal of Pathology

Article Title: Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

doi: 10.1016/j.ajpath.2012.09.004

Figure Lengend Snippet: Effect of PARP-1 deficiency on histopathology. PARP-1–deficient and WT mice were treated with bleomycin to induce lung injury or with saline as a control. On day 21 after treatment, lung tissue sections were obtained and stained with Masson’s trichrome. Representative sections from WT mice treated with saline (A and E) or bleomycin (B and F) and from PARP-1 KO mice treated with saline (C and G) or bleomycin (D and H) show reduced fibrosis in the KO mice. Original magnification, ×100 (A–D); ×400 (E–H).

Article Snippet: Selected samples were pretreated with high-specific-activity PARP-1 enzyme (4668-100-01; Trevigen, Gaithersburg, MD) in 1× PARP buffer (4671-096-02; Trevigen) containing the NAD + substrate of PARP-1 before addition of the 32 P-labeled probe (20,000 to 30,000 counts/minute).

Techniques: Histopathology, Saline, Staining

Journal: The American Journal of Pathology

Article Title: Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

doi: 10.1016/j.ajpath.2012.09.004

Figure Lengend Snippet: Effect of PARP-1 deficiency on lung fibroblasts. A: Lung fibroblasts isolated from either saline- or bleomycin-treated WT or PARP-1–deficient mice (7 days after bleomycin treatment) were analyzed for α-SMA and α1(I) procollagen proteins by Western blotting. Each lane represents a sample from a single animal. GAPDH was used as a loading control, after stripping of the membrane. B: Relative density of the bands was quantified from three independent experiments, normalized to the density of corresponding GAPDH bands. Data are expressed as means ± SEM. n = 3. *P < 0.05.

Article Snippet: Selected samples were pretreated with high-specific-activity PARP-1 enzyme (4668-100-01; Trevigen, Gaithersburg, MD) in 1× PARP buffer (4671-096-02; Trevigen) containing the NAD + substrate of PARP-1 before addition of the 32 P-labeled probe (20,000 to 30,000 counts/minute).

Techniques: Isolation, Saline, Western Blot, Stripping Membranes, Membrane

Journal: The American Journal of Pathology

Article Title: Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

doi: 10.1016/j.ajpath.2012.09.004

Figure Lengend Snippet: PARP-1 expression is up-regulated in human fibrotic lung fibroblasts and fibroblastic foci. A: Lung fibroblasts from IPF patients or from control subjects (CON) without pulmonary fibrosis were analyzed for PARP-1 and α-SMA mRNAs by qPCR. 18S rRNA was used as the reference, and the level in cells from control subjects (CON) was used as the calibrator. B: Lung fibroblasts from IPF patients or from control subjects (CON) without pulmonary fibrosis were analyzed for PARP activity in the presence or absence of 3-AB (10 mmol/L). C: Lung tissue sections from two IPF patients (top panels) and a control subject (bottom left panel) were stained with anti–PARP-1 (nickel; black stain) and α-SMA (NovaRED; red stain) antibodies. The IPF lung sections show fibroblastic foci with cells positively staining for both nuclear PARP-1 and cytoplasmic α-SMA (arrows). The control lung section shows no detectable staining for PARP-1, but shows positive staining for α-SMA in airway and vascular smooth muscle cells. Nonimmune IgG control (bottom right panel) staining of an IPF lung section confirmed specificity of staining. Data are expressed as means ± SEM. n = 3 (A); n = 5 (B). *P < 0.05 versus control. Scale bar = 50 μm. Original magnification, ×400.

Article Snippet: Selected samples were pretreated with high-specific-activity PARP-1 enzyme (4668-100-01; Trevigen, Gaithersburg, MD) in 1× PARP buffer (4671-096-02; Trevigen) containing the NAD + substrate of PARP-1 before addition of the 32 P-labeled probe (20,000 to 30,000 counts/minute).

Techniques: Expressing, Activity Assay, Staining