Journal:

Article Title: Phosphorylation at Carboxyl-Terminal S373 and S375 Residues and 14-3-3 Binding Are Not Required for Mouse p53 Function 1

doi:

Figure Lengend Snippet: Mouse p53 phosphomutants are not defective in inducing cell-cycle arrest. (A) Asynchronous p53-/- MEFs were transfected with 5 µg of empty vector, wild-type p53 constructs, or phosphomutant p53 constructs, and were γ-irradiated at 20 Gy. Simultaneous staining of intracellular p53 (with Pab246 primary antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG antibody) and genomic DNA (by propidium iodide) was performed before analysis by flow cytometry. p53-expressing cells, which emitted green fluorescence, were gated (except for vector-transfected controls), and their cell-cycle profile was determined by ModFIT cell-cycle analysis software (BD Biosciences). (B) Cell death was analyzed by determining sub-G1 populations in cell-cycle analysis, as described in (A), by flow cytometry. Cells treated with and without 20 Gy γ-irradiation and harvested 24 hours after γ-irradiation were analyzed. The experiment was performed thrice independently. Data from three independent experiments are presented as mean ± SD. (C) p53-/- MEFs were cotransfected with pcDNA-based constructs, and cells were selected with 2 µg/ml puromycin (Sigma) for 10 to 15 days and stained with crystal violet (Sigma). Colony formation assay was performed four independent times, and representative data are shown.

Article Snippet: Simultaneous staining of intracellular p53 (with Pab246 primary antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG antibody) and genomic DNA (by propidium iodide) was performed as described [ 19 ], and the cells were analyzed by flow cytometry with FACSCaliber (BD Biosciences, Franklin Lakes, NJ). p53-expressing cells, which emitted green fluorescence, were gated (except for vector-transfected controls), and their cell-cycle profile was determined by ModFIT cell-cycle analysis software (BD Biosciences).

Techniques: Transfection, Plasmid Preparation, Construct, Irradiation, Staining, Flow Cytometry, Expressing, Fluorescence, Cell Cycle Assay, Software, Colony Assay