polyclonal antipolymer  (Aparptosis Inc)

Journal:

Article Title: Functional Interaction between Poly(ADP-Ribose) Polymerase 2 (PARP-2) and TRF2: PARP Activity Negatively Regulates TRF2

doi: 10.1128/MCB.24.4.1595-1607.2004

Figure Lengend Snippet: PARP-2 interacts with TRF2. (A) Lysates from Cos1 cells expressing Myc-hTRF2 fusion protein (lanes 1 to 5) together with either GST (lane 1) or GST-mPARP-2 fusion protein (lanes 2 to 5) were analyzed by GST pull down under increasing stringency conditions of washing buffers as indicated, followed by Western blotting using, successively, anti-Myc (top) and anti-GST (bottom) antibodies. (B) Conditions of interaction between PARP-2 and TRF2. Interaction of GST (lane 1) or GST-mPARP-2 fusion protein (lanes 2 to 5) with Myc-hTRF2 fusion protein (lanes 1 to 5) in Cos1 cells either untreated (lane 2) or treated with 4 mM N-nitroso-N-methylurea (lane 3), 2 mM 3-AB (lane 4), or 10 μg of ethidium bromide/ml (lane 5). Proteins were analyzed by GST pull down and Western blotting using, successively, anti-Myc, anti-GST, and antipolymer antibodies as indicated.

Article Snippet: Blots were subsequently incubated with mouse monoclonal anti-Myc antibody (1/250; 9E10; Santa Cruz Biotechnology), mouse monoclonal anti-GST antibody (1/10,000; IGBMC, Illkirch, France), and rabbit polyclonal antipolymer antibody (1/1,000; Biomol Research Labs).

Techniques: Expressing, Western Blot

Journal:

Article Title: Functional Interaction between Poly(ADP-Ribose) Polymerase 2 (PARP-2) and TRF2: PARP Activity Negatively Regulates TRF2

doi: 10.1128/MCB.24.4.1595-1607.2004

Figure Lengend Snippet: Colocalization of PARP-2 and TRF2 in U2OS cells. U2OS cells were transfected with GFP-mPARP-2 proteins (green), fixed 13 h postrelease of a double thymidine block, and stained red with Alexa 568-labeled rabbit anti-PML antibody (a to c) or Alexa 594-labeled goat anti-TRF2 antibody (d to i). To detect PARP activity, cells were treated with 5 mM H2O2 for 10 min before fixation and costained with Alexa 594-labeled anti-TRF2 (red) and fluorescein isothiocyanate-conjugated antipolymer antibodies (green) (g to i).

Article Snippet: Blots were subsequently incubated with mouse monoclonal anti-Myc antibody (1/250; 9E10; Santa Cruz Biotechnology), mouse monoclonal anti-GST antibody (1/10,000; IGBMC, Illkirch, France), and rabbit polyclonal antipolymer antibody (1/1,000; Biomol Research Labs).

Techniques: Transfection, Blocking Assay, Staining, Labeling, Activity Assay

Journal:

Article Title: Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage

doi: 10.1073/pnas.012460399

Figure Lengend Snippet: MNNG-induced PARP activation and acidification in Molt 3 cells. (A) Polymer immunoblot. Molt 3 cells were treated with 10 or 100 μM MNNG for a given time and immunoblotted with anti-polymer LP96–10. The blot represents one of the four experiments with identical results. (B) NAD and ATP depletion. Samples of Molt 3 cells, treated with 10 or 100 μM MNNG as above, were analyzed for NAD (○) or ATP (□). Results (mean ± SD) were obtained from four experiments, each in triplicate. (C) Time course of acidification. Molt 3 cells were treated with 10 (◊) or 100 (■) μM MNNG, and changes in pH were monitored by BCECF method up to 7 h. Results (mean ± SD) were obtained from four experiments, each in triplicate.

Article Snippet: Immunoblotting was carried out as described ( 22 ) with monoclonal anti-PARP (C-2–10, 1:10,000, Aparptosis), polyclonal antipolymer (LP96–10, 1:10,000, Aparptosis), monoclonal antipolymer ( 25 ) (10H, 1:1,000), or polyclonal anticaspase 3 (3-R#MF393 from D. Nicholson, Merck Frosst Canada, Montreal, 1:10,000).

Techniques: Activation Assay, Western Blot

Journal:

Article Title: Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage

doi: 10.1073/pnas.012460399

Figure Lengend Snippet: Role of PARP in acidification response. (A) Suppression of PARP activation with DHQ. Molt 3 cells were exposed to 100 μM MNNG after 5-min pretreatment with 100 μM DHQ and immunoblotted for polymer with LP96–10. This blot represents one of the four experiments with identical results. (B) Suppression of acidification with PARP inhibitor. BCECF-loaded Molt 3 cells were exposed to 100 μM MNNG or 300 μM H2O2 with or without 5-min pretreatment with 100 μM DHQ, and changes in pH were measured at 30 min. Results (mean ± SD) were obtained from four experiments, each in triplicate. (C) MNNG-induced polymer synthesis in PARP+/+ and PARP−/− fibroblasts. Cells with two PARP genotypes were treated with 300 μM MNNG and immunoblotted with anti-polymer LP96–10. This blot represents one of the three experiments with identical results. (D) MNNG-induced acidification in PARP+/+ and PARP−/− fibroblasts. The pH changes in BCECF-loaded cells were measured at 30 min after exposure to 300 μM MNNG. Results (mean ± SD) were obtained from two experiments, each in triplicate.

Article Snippet: Immunoblotting was carried out as described ( 22 ) with monoclonal anti-PARP (C-2–10, 1:10,000, Aparptosis), polyclonal antipolymer (LP96–10, 1:10,000, Aparptosis), monoclonal antipolymer ( 25 ) (10H, 1:1,000), or polyclonal anticaspase 3 (3-R#MF393 from D. Nicholson, Merck Frosst Canada, Montreal, 1:10,000).

Techniques: Activation Assay

Journal:

Article Title: Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage

doi: 10.1073/pnas.012460399

Figure Lengend Snippet: Impact of acidification on mode of cell death. (A) PARP activation in pH-clamped cells. Molt 3 cells were treated for 60 min with 10 μM MNNG without pH clamp, as in Fig. ​Fig.33A, or with pH 6.8 clamp (lanes 1–5). Another set of cells was treated with 100 μM MNNG without pH clamp, as in Fig. ​Fig.33A, or with pH 7.4 clamp (lanes 6–10). Samples were immunoblotted with antipolymer LP96–10. (B) Flow cytometry analysis of mode of cell death. Cells treated for 1 h with 10 or 100 μM MNNG with or without pH clamp were allowed to recover for 10 h, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry. The viable cells were identified by low signals for both the dyes, whereas apoptotic cells were detected by exclusion of propidium iodide and staining with annexin V. In contrast, necrotic cells were detected by high uptake of both the dyes. (C) Caspase 3-immunoblot analysis of cell death. Cells treated as described in B were immunoblotted for caspase 3. All lanes marked C represent DMSO-treated controls, and etoposide-treated HL-60 cells were used as positive apoptosis control in both C and D (lane 9). (D) PARP immunoblot analysis of cell death. Cells treated as described in B were also immunoblotted for PARP. All data represent one of three experiments with identical results.

Article Snippet: Immunoblotting was carried out as described ( 22 ) with monoclonal anti-PARP (C-2–10, 1:10,000, Aparptosis), polyclonal antipolymer (LP96–10, 1:10,000, Aparptosis), monoclonal antipolymer ( 25 ) (10H, 1:1,000), or polyclonal anticaspase 3 (3-R#MF393 from D. Nicholson, Merck Frosst Canada, Montreal, 1:10,000).

Techniques: Activation Assay, Flow Cytometry, Staining, Western Blot