Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Mitochondrial-localized NADPH oxidase 4 is a source of superoxide in angiotensin II-stimulated neurons

doi: 10.1152/ajpheart.00974.2012

Figure Lengend Snippet: Immunofluorescence reveals Nox4 localized to neuron mitochondria. A : high-magnification confocal microscopy image showing punctate distribution of Nox4 (arrows) in fixed CATH.a neurons incubated with anti-Nox4 primary antibody followed by an Alexa Fluro 594 (red fluorescence) secondary antibody. Scale bar = 10 μm. B : confocal microscopy images showing colocalization (yellow fluorescence in merged image) of Nox4 (red) and the mitochondrial protein MnSOD (green). Scale bar = 20 μm. C : confocal microscopy images showing colocalization (yellow fluorescence in merged image) of Nox4 (green) and MitoTracker Red. Scale bar = 10 μm.

Article Snippet: Neurons were washed with 0.1 mol/l PB, and then incubated with Alexa Fluro 488 or 594 secondary antibody (1:500, anti-rabbit, Invitrogen, Carlsbad, CA) at room temperature for 2 h. After washout of secondary antibody, fluorescent images were captured by confocal laser scanning microscopy (Zeiss LSM 510 Meta).

Techniques: Immunofluorescence, Confocal Microscopy, Incubation, Fluorescence

Journal: Nucleic Acids Research

Article Title: hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity

doi: 10.1093/nar/gkq1340

Figure Lengend Snippet: Mapping of region in hSSB1 required to pull-down MRN complex. ( a ) Pull-down reactions were performed by incubating full-length hSSB1 or C-terminal truncations of hSSB1 (T1 1–153, T2 1–114) bound to sepharose beads with purified recombinant MRN (20 ng) complex. After washing, beads were subjected to immunoblotting with indicated antibodies. Coomassie stained gel shows the relative input of hSSB1 protein. ( b ) hSSB1 tail (amino acids 153–211) is sufficient for interaction with Mre11 in nuclear extracts. Cells were transfected with GFP-tagged hSSB1-tail or GFP vector alone and anti-GFP immunoprecipitates were immunoblotted with indicated antibodies. ( c ) Overexpression of GFP-hSSB1-tail (MRN interacting domain) results in impaired Mre11 foci formation after IR. HeLa cells were transfected with GFP or GFP-hSSB1-tail. Forty-eight hours after transfection cells were treated with IR (6 Gy, 30 min) and immunostained with indicated antibodies. Overexpression of GFP-hSSB1 tail (MRN interacting domain) results in impaired Mre11 foci formation after IR. HeLa cells were transfected with GFP or GFP-hSSB1-tail. Forty-eight hours after transfection cells were treated with IR (6 Gy, 30 min) and immunostained with indicated antibodies. ( d ) Overexpression of GFP-hSSB1 tail (MRN interacting domain) abrogates the IR induced G1/S checkpoint. HeLa cells were transfected with GFP or GFP-hSSB1-tail (GFP-CT). Forty-eight hours after transfection cells were treated with IR (6 Gy, 30 min) and allowed to recover for 16 h after which cells were pulse labeled with with BrdUrd (30 min, 10 μg/ml). Cells were subsequently stained with anti-BrdUrd followed by Alexa 488 secondary antibodies and propidium iodide before being analysed by flow cytometry. The box illustrating BrdUrd positive cells indicates that GFP-transfected cells effectively arrest at the G1/S boundary compared to GFP-CT transfected cells, which continue to enter S-phase.

Article Snippet: Antibodies The following antibodies were used in the study: Rad50 (Calbiochem), Mre11, NBS1 (Sigma), IntS3 (Bethyl laboratories) and Alexa secondary antibodies (Invitrogen).

Techniques: Purification, Recombinant, Staining, Transfection, Plasmid Preparation, Over Expression, Labeling, Flow Cytometry, Cytometry