Computational protocol: The centrosomal OFD1 protein interacts with the translation machinery and regulates the synthesis of specific targets

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Protocol publication

[…] HEK 293 cells and kidneys from fed animals were homogenized in lysis buffer (Tris 50 mM pH 7.9, 1% triton X-100, 0.1% Tween20, 150 mM NaCl, 10% glycerol, 5 mM MgCl2) or in a specific buffer for Co-IP experiments (50 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2, 5 mM EGTA, 0.5% Triton X-100, pH 7.28). For Co-IP experiments, lysates were incubated with specific antibodies and IgG, as control, as described. Co-IP experiments were performed at least three times. Western blot (WB) studies were performed at least in triplicate and representative images were shown. For Co-IP experiments the ratio of IP proteins with respect to the input was 1:50. Blots were quantified by ImageJ. The control was settled as 1 and the fold change was calculated and reported below the panels as the mean ± standard error of the mean (SEM). Animals were perfused with PBS to eliminate blood traces for detection of the GH protein. Lysates were treated with protease inhibitors from Sigma-Aldrich (P8340) and phosphatase inhibitors from Roche (PhosSTOP, 04906837001). Polyvinylidene difluoride (PVDF) membranes were used for Immunoblot (Millipore, US, Immobilon-P, IPVH00010) and ECL western blotting reagent (Thermoscientific, 32106) or Femto (Thermoscientific, 34095) were used for detection. [...] Cells were fixed in methanol or in PFA 4%. Blocking was performed in PBS 0.2% TritonX-100, 10% FBS. IF experiments were performed at least three times. For analysis of IF data more than 100 cells were counted for each experiment. The significance of the results was calculated by Student’s t-test and reported as pvalue. In IF experiments, co-localization at the centrosome were considered biological relevant when present in >50% of cells.For the colocalization analysis at the centrosome, we selected the centrosomal area defined by γtubulin signal and measured the fluorescence signal intensity of the protein/mRNA of interest. An equal area was selected in three different positions in the cell and the average value was calculated and considered as the mean fluorescence intensity of the cell. Cells in which the signals were considered to colocalize were characterized by a higher fluorescence signal at the centrosome compared to the mean fluorescence intensity of the cell. Fluorescence intensity was calculated by ImageJ.High-resolution confocal microscopy “LSM 880/Elyra PS-1” Zeiss with superresolution structured illumination processing was used to obtain high-resolution images. […]

Pipeline specifications

Software tools ImageJ, Coloc
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Kidney Diseases