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

Journal: Nucleic Acids Research

Article Title: Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins modulates splicing

doi: 10.1093/nar/gkp218

Figure Lengend Snippet: Reduced RNA bound to hnRNPs by pADPr and PARG loss-of-function. ( A ) pADPr inhibits human GST-hnRNPA1 binding to RNA, as shown by EMSA. 25 fmol biotin-labeled hnRNPA1 binding sequence was incubated with the components as indicated: 1 . Control; 2 . 100 ng GST; 3 . 100 ng human GST-hnRNP A1; 4 . 100 ng human GST-hnRNP A1 preincubated with 70 ng pADPr; 5 . 100 ng human GST-hnRNP A1 preincubated with 140 ng pADPr. *The trace of higher molecular weight RNA is present in all reactions, which likely comes from the synthetic RNA pool. ( B ) Reduced RNA bound to hrp38:GFP by PARG loss-of-function. The amount of hsrω-RA (spliced product) co-immunoprecipitated with hrp38:GFP as a percent of the input was measured by real-time RT–PCR: 1 . hrp38:GFP strain immunoprecipitated with IgG as a control; 2 . hrp38:GFP strain immunoprecipitated with anti-GFP; 3 . Parg27.1; hrp38:GFP strain immunoprecipitated with anti-GFP. The error bar represents the standard deviation from two independent experiments (** P ≤ 0.01).

Article Snippet: For pADPr inhibition assay, 100 ng human GST-hnRNPA1 was preincubated with 70 ng or 140 ng pADPr (Biomol) in 1× binding buffer for 20 min at 25°C.

Techniques: Binding Assay, Labeling, Sequencing, Incubation, Control, Molecular Weight, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation