nb 100 182 rrid ab 350110  (Novus Biologicals)

Journal: PLoS ONE

Article Title: Ouabain, a Cardiac Glycoside, Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

doi: 10.1371/journal.pone.0075905

Figure Lengend Snippet: (A) The data from chemical library screening and chemical structure of ouabain. U2OS cells were pre-treated with 5 µM of each compound for 1 h prior to incubation in 200 ng/ml MMC for 24 h. Representative data from plate containing ouabain compound are shown. Each dot in the scattergram represents FANCD2 foci area per cell after treatment with each compound tested. (B) Ouabain inhibits MMC-induced FANCD2 foci formation. U2OS cells were pretreated with the indicated concentration of ouabain or curcumin for 1 h and then treated with 200 ng/ml MMC for 24 h. After incubation, the cells were fixed and processed for FANCD2 immunofluorescence and the FANCD2 foci were analyzed with an IN Cell Analyzer. Representative graphs and images from three independent experiments are shown. Values represent the means ± SEM (Student’s t -test, ***, P < 0.001; *, P < 0.05). (C) U2OS cells treated as described in (B) were analyzed with an IN Cell Analyzer. % foci total area/ cell represents relative % to MMC-only treated samples. IC 50 was calculated using GraphPad Prism version 5.03. Graphs and values represent the means ± SEM from three independent experiments. (D) Ouabain inhibits MMC-induced FANCD2 monoubiquitination. Protein extracts fromU2OS cells treated as described in (B) were analyzed by Western blotting using antibodies against FANCD2 and β-actin. Values shown represent the ratio of FANCD2 (L)/FANCD2 (S). (E) Ouabain inhibits FANCD2 monoubiquitination after cisplatin treatment. Western blotting was performed with protein extracts from U2OS cells treated with 10 µM cisplatin.

Article Snippet: Antibodies against FANCD2 (NB 100-182) and FANDI (A300-212A) were purchased from NOVUS (Littleton, CO) and Bethyl Laboratories (Montgomery, TX), respectively.

Techniques: Library Screening, Incubation, Concentration Assay, Immunofluorescence, Western Blot

Journal: PLoS ONE

Article Title: Ouabain, a Cardiac Glycoside, Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

doi: 10.1371/journal.pone.0075905

Figure Lengend Snippet: (A) U2OS cells were pretreated with the indicated concentration of digitoxin or digoxin for 1 h and then incubated in 200 ng/ml MMC for 24 h. After incubation, the cells were fixed and processed for FANCD2 immunofluorescence and the FANCD2 foci were analyzed with IN Cell Analyzer. Representative graphs from three independent experiments are shown. Values represent the means ± SEM.(B) (C) Protein extracts from U2OS cells pretreated with 5 µM curcumin, 100 nM ouabain, 100 nM digitoxin and 100 nM digoxin and incubated in 200 ng/ml MMC for 24 h as described in (A) were analyzed by Western blotting using antibodies against (B) FANCD2, (C) FANCI, and β-actin. L/S values shown represent the ratio of FANCD2 (L)/FANCD2 (S) or FANCI (L)/FANCI (S).

Article Snippet: Antibodies against FANCD2 (NB 100-182) and FANDI (A300-212A) were purchased from NOVUS (Littleton, CO) and Bethyl Laboratories (Montgomery, TX), respectively.

Techniques: Concentration Assay, Incubation, Immunofluorescence, Western Blot

Journal: PLoS ONE

Article Title: Ouabain, a Cardiac Glycoside, Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

doi: 10.1371/journal.pone.0075905

Figure Lengend Snippet: (A) Protein extracts from U2OS cells treated with 100 nM ouabain for the indicated times were analyzed by Western blotting using antibodies against phosphorylated p38, p38, phosphorylated ERK, ERK, phosphorylated JNK, and JNK. (B) U2OS cells were pretreated with the indicated concentration of MAPK inhibitors for 30 min and then with 100 nM ouabain for 30 min and incubated in 200 ng/ml MMC for 24 h. The cell lysates were subjected to Western blot analysis using antibodies to FANCD2 and β-actin. PD, PD 98059 (ERK inhibitor); SP, SP 600215 (JNK inhibitor); SB, SB 203580 (p38 inhibitor).

Article Snippet: Antibodies against FANCD2 (NB 100-182) and FANDI (A300-212A) were purchased from NOVUS (Littleton, CO) and Bethyl Laboratories (Montgomery, TX), respectively.

Techniques: Western Blot, Concentration Assay, Incubation

Journal: Oncogene

Article Title: Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation.

doi: 10.1038/sj.onc.1207895

Figure Lengend Snippet: Figure 4 Depletion of hSNM1B by siRNA does not affect monoubiquitination of FANCD2. SV40-transformed fibroblasts (GM0637) were transfected with hSNM1B, FANCA or a negative control siRNA and assayed 66 h later for monoubiquitination of FANCD2 by immunoblot. Cells were either untreated or treated with MMC or IR as indicated

Article Snippet: Polyclonal Nibrin/p95 (NB100-143) and FANCD2 (NB 100- 182) antibodies were purchased from Novus-Biologicals (Littleton, CO, USA).

Techniques: Transformation Assay, Transfection, Negative Control, Western Blot

Journal: Oncogene

Article Title: Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation.

doi: 10.1038/sj.onc.1207895

Figure Lengend Snippet: Figure 3 Depletion of hSNM1B from HeLa cells increases sensitivity to ICL-inducing agents and to IR. (a) Clonogenic survival of HeLa cells transfected with hSNM1B siRNA, FANCD2 siRNA or control siRNA after treatment with increasing concentrations of MMC. (b) Survival of hSNM1B-depleted and control siRNA-transfected cells after treatment with cisplatin or (c) survival of HeLa cells transfected with hSNM1B siRNA, 53BP1 siRNA or control siRNA after treatment with increasing doses of IR. A fraction of HeLa cells treated with siRNAs for use in survival experiments was assayed for protein expression. Whole-cell lysates were immunoblotted for FANCD2 (d) or for 53BP1 (e). p95/nibrin expression was assayed as a control for specificity of RNA interference

Article Snippet: Polyclonal Nibrin/p95 (NB100-143) and FANCD2 (NB 100- 182) antibodies were purchased from Novus-Biologicals (Littleton, CO, USA).

Techniques: Transfection, Control, Expressing