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

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Figure Lengend Snippet: Steady-state level and transactivation ability of mouse p53 phosphomutants. (A) The alignment of the C-termini of human (accession no. NP_000537) and mouse (accession no. NP_035770) p53 proteins was performed by the Web-based alignment program BLAST2 (National Center for Biotechnology Information). Conserved residues are shown in the middle row between human and mouse p53 sequences. Serine residues at positions 375 and 378 for human p53, and at positions 373 and 375 for mouse p53, are indicated by boxes. (B) The S373 and S375 residues of mouse p53 were mutated to nonphosphorylatable alanine (A) residues or aspartic acid (D) that mimics phosphorylated residues, and were expressed in pCDNA or pBabe vectors. p53-/- MEFs were cotransfected with 1 µg of empty vector, wild-type or mutant mouse p53, and 0.2 µg of GFP-encoding plasmids, and the steady-state level of p53 phosphomutants was determined by Western blot analysis. (C) The transcriptional activity of the mouse phosphomutants was determined by luciferase assays, using mdm2 promoter-luciferase construct. Luciferase activity was determined by chemiluminescence and was normalized against β-galactosidase activity. The experiment was performed thrice independently. Data from one of the experiments, performed in duplicate, are presented as mean ± SD. (D) Relative luciferase activity using the mdm2 promoter-luciferase construct was determined in mouse p53-transfected γ-irradiated (10 Gy) p53-/- MEFs, as described above. The experiment was performed twice independently. Data from one of the experiments, performed in duplicate, are presented as mean ± SD. (E and F) Relative luciferase activity using p21 (E) and mdm2 promoter-luciferase constructs (F) together with human wild-type or phosphomutant IND constructs in p53-null H1299 cells. Data from one of the experiments, performed in duplicate, are presented as mean ± SD. (G and H) The expression of endogenous p53 target genes (bax, noxa, mdm2, and p21) in vector-transfected and p53-transfected p53-/- MEFs (G) or human H1299 cells (H) was determined by semiquantitative RT-PCR and normalized against endogenous gapdh RNA level as described previously.

Article Snippet: All subsequent experiments described were performed with p53 mutants based on both types of vectors, which showed similar and consistent results. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 Steady-state level and transactivation ability of mouse p53 phosphomutants. (A) The alignment of the C-termini of human (accession no. NP_000537) and mouse (accession no. NP_035770) p53 proteins was performed by the Web-based alignment program BLAST2 (National Center for Biotechnology Information).

Techniques: Plasmid Preparation, Mutagenesis, Western Blot, Activity Assay, Luciferase, Construct, Transfection, Irradiation, Expressing, Reverse Transcription Polymerase Chain Reaction