Computational protocol: 8p22 MTUS1 Gene Product ATIP3 Is a Novel Anti-Mitotic Protein Underexpressed in Invasive Breast Carcinoma of Poor Prognosis

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Protocol publication

[…] Exponentially growing cells plated on coverslips were fixed in ice-cold methanol for 5 min prior to incubation for 1 hr at room temperature with primary antibodies. SK-MES cells were incubated with human anti-alpha-tubulin antibodies clone F2C diluted 1∶10 and mouse anti-MTUS1 monoclonal antibodies diluted 1∶10 (Abnova, clone 1C7). HeLa, MDA-MB-231 or MCF7 cells transiently transfected (24 hrs) with pEGFP-C1 or GFP-ATIP3 were incubated with mouse anti-alpha-tubulin monoclonal antibodies (Sigma) diluted 1∶500. After extensive washing, cells were incubated for 30 min at room temperature with appropriate secondary Cy-2-conjugated anti-human and/or Cy-3-conjugated anti-mouse antibodies (Jackson Laboratories, Interchim, France) diluted 1∶500. Coverslips were mounted on glass slides using Glycergel Mounting Medium (DakoCytomation) and examined with a Leica TCS SP2 AOBS confocal microscope using appropriate filters. Image analysis was performed with NIH ImageJ software.For live cell imaging, images were acquired on a spinning disk microscope (one image taken every 5 min for 36 hrs following transient transfection of HeLa-mCherryH2B cells) as described . Multi-dimensional acquisitions were performed in time-lapse mode using Metamorph 7.1.7 software. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications SPIM, Microscopic phenotype analysis
Diseases Breast Neoplasms, Colonic Neoplasms, Neoplasms