Journal: iScience

Article Title: SART1 modulates poly-(ADP-ribose) chain accumulation and PARP1 chromatin localization

doi: 10.1016/j.isci.2024.111252

Figure Lengend Snippet:

Article Snippet: Mouse anti-PAR antibody , RD-Systems , 4335-MC-100.

Techniques: Virus, Recombinant, Lysis, Extraction, Protease Inhibitor, Transfection, Mutagenesis, Western Blot, Purification, Expressing, Plasmid Preparation, Software, Microscopy

Journal: Genetics

Article Title: A Context-Dependent Role for the RNF146 Ubiquitin Ligase in Wingless/Wnt Signaling in Drosophila

doi: 10.1534/genetics.118.301393

Figure Lengend Snippet: Drosophila Rnf146/Iduna targets Tnks substrates for degradation in vivo. (A) Schematic representation of the domain structure of RNF146. Residues in human RNF146 and the Drosophila homolog that are identical are shown in red. All residues in the human RNF146 WWE domain that bind poly-ADP-ribose are identical in Drosophila Rnf146 and indicated with asterisks. (B) Schematic representation of Drosophila Rnf146 genomic region and deletions in two Rnf146 mutants. Insertion site of the P element EY09040 is indicated (green). A fragment of the P element EY09040 remains in the Rnf146157 mutant. (C) Lysates from wild-type and Rnf146 mutant larvae were analyzed by immunoblot using pADPr antibody. Tubulin was used as a loading control. The levels of poly-ADP-ribosylated proteins are increased in Rnf146 null mutant third-instar larvae, but revert to baseline in Rnf146 Tnks double null mutants. (D–I) Third-instar larval wing imaginal discs with Rnf14636 null mutant clones marked by the absence of GFP (−/−; magenta) were stained with indicated antibodies. Proteins modified by poly-ADP-ribose (D) accumulate cell autonomously in Rnf14636 mutant clones but revert to baseline upon concomitant inactivation of Tnks (Tnks19) (G–I). Blue is the merge of magenta and yellow.

Article Snippet: The primary antibodies used for immunostaining were mouse anti-V5 (1:5000; Invitrogen), mouse anti-Wingless (1:200, 4D4 concentrated antibody; Developmental Studies Hybridoma Bank, DSHB), guinea pig anti-Senseless (1:1000) ( Nolo et al. 2000 ), rabbit anti-β-gal (1:1000; MP Biomedicals), mouse anti-pADPr (1:1000, clone 10H; Tulip Biolabs), rabbit anti-GFP (1:200; Invitrogen).

Techniques: In Vivo, Mutagenesis, Western Blot, Clone Assay, Staining, Modification

Journal: Nucleic Acids Research

Article Title: Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

doi: 10.1093/nar/gkn771

Figure Lengend Snippet: SDS–PAGE analysis of pADPr-associated proteins from MNNG-treated and PARG-silenced SK-N-SH cells after immunoprecipitation with anti-pADPr antibodies. pADPr-associated proteins were immunoprecipitated using anti-pADPr mouse monoclonal antibody clone 10H bound to Protein G coated magnetic beads. Immunoprecipitates were resolved by 4–12% SDS–PAGE and stained with SYPRO Ruby fluorescent dye. Normal mouse IgGs were used to assess nonspecific binding. Selected proteins identified by LC-MS/MS, mostly involved in DNA/RNA transactions, are shown (see Supplementary Table S3 for complete protein listing).

Article Snippet: The Dynabeads™ were washed two times with 1 ml of 0.1 M sodium acetate buffer, pH 5.0, coated with 10–15 μg of mouse monoclonal anti-pADPr antibody clone 10H (Tulip Biolabs, West Point, PA, USA) or equivalent amount of normal mouse IgGs (Calbiochem-EMD Biosciences, San Diego, CA, USA).

Techniques: SDS Page, Immunoprecipitation, Magnetic Beads, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Nucleic Acids Research

Article Title: Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

doi: 10.1093/nar/gkn771

Figure Lengend Snippet: Validation of selected pADPr-associated proteins identified by LC-MS/MS using western blot analysis of pADPr immunoprecipitates. The specificity of the pADPr immunoprecipitation using anti-pADPr 10H monoclonal antibodies was evaluated by immunoblot analysis as described in Materials and methods section. The same proteins were not precipitated by normal mouse IgGs, confirming the specificity of the pull-down.

Article Snippet: The Dynabeads™ were washed two times with 1 ml of 0.1 M sodium acetate buffer, pH 5.0, coated with 10–15 μg of mouse monoclonal anti-pADPr antibody clone 10H (Tulip Biolabs, West Point, PA, USA) or equivalent amount of normal mouse IgGs (Calbiochem-EMD Biosciences, San Diego, CA, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Immunoprecipitation

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