Computational protocol: TRAIP regulates replication fork recovery and progression via PCNA

Similar protocols

Protocol publication

[…] U2OS cells were sequentially labeled with IdU (50 μm) and CidU (100 μm). Thereafter, labeled cells were harvested and DNA fiber spreads were prepared on silanized slides (DAKO, Santa Clara, CA, USA) by addition of 200 mm Tris-HCl pH 7.5, 50 mm EDTA, 0.5% SDS []. We fixed and denatured DNA fibers using methanol/acetic acid (3:1), and 2.5 m HCl, respectively. The acid-treated fiber spreads were subsequently neutralized with Borax buffer (0.1 m borax, pH 8.5), and co-stained with mouse anti-BrdU antibodies (BD Biosciences, clone B44; diluted at 1:100) and rat anti-BrdU antibodies (AbD Serotec, Hercules, CA, USA, clone BU1/75; diluted at 1:100). Secondary antibodies were Rhodamine goat anti-mouse IgG and FITC goat anti-rat IgG (both from Jackson ImmunoResearch Laboratories, West Groves, PA, USA; diluted at 1:400). Fibers were examined under fluorescence microscope. For quantification of replication structures, at least 250 structures were counted per experiment. The relative lengths of red (Rhodamine) and green (FITC) labeled patches were measured using the ImageJ software (National Institutes of Health; http://rsbweb.nih.gov/ij). [...] H2B-GFP expressing HeLa cells were seeded, and were transfected with control or TRAIP siRNAs. Twenty-four hours post transfection cells were placed in a live cell stage-mounted environment chamber and images were captured at 5 min intervals using Perkin Elmer Spinning Confocal Microscope (Waltham, MA, USA) for 8 h. Data were analyzed using the MetaMorph analysis software (Sunnyvale, CA, USA). […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens