Journal: The Journal of Biological Chemistry
Article Title: Cancer Susceptibility Polymorphism of p53 at Codon 72 Affects Phosphorylation and Degradation of p53 Protein *
doi: 10.1074/jbc.M110.208587
Figure Lengend Snippet: Degradation of p53-72P by Mdm2 is accelerated compared with p53-72R. A, pcDNA3-p53-72P or -72R together with N-terminally c-Myc-tagged Mdm2 (pCMV-Myc-Mdm2; Mdm2 expressed from a CMV promoter) or control empty vector were introduced into H1299 cells (4.4 × 105 cells/10-cm dish) and analyzed by Western blotting. 0.44 μg of p53 and 4 μg of Mdm2 were transfected. Cells were harvested 21 h post-transfection. Levels of p53 (normalized by β-actin) were quantified and are shown below the panels. B, pcDNA3-p53-72P or -72R (0.35 μg) together with His6-tagged ubiquitin (2.2 μg) and N-terminally FLAG-tagged Mdm2 (pSG-FLAG-Hdm2, 1.42 μg) or control empty vector (vec) (1.42 μg) were introduced into H1299 cells (6 × 105 cells/10-cm dish), and cells were harvested 27 h post-transfection. To detect ubiquitinated p53 efficiently, Mdm2 was expressed at a low level using expression plasmid pSG-F-Hdm2 (Hdm2 is under the control of SV40 promoter, which is much weaker than CMV promoter). p53 was immunoprecipitated (IP) with anti-p53 polyclonal antibody (FL393), and immunoprecipitated samples and whole cell lysates (WCL) were analyzed by Western blotting. Western blot analyses of immunoprecipitates were performed with the anti-His antibody to detect ubiquitinated p53 (upper panel) or with FL393 antibody to detect nonubiquitinated p53 (lower panel). Levels of ubiquitinated p53 (normalized by nonubiquitinated p53) were quantified and are shown below the panels. C, pMX-p53-72P or -72R (0.5 μg) together with pCMV-Myc-Mdm2 (4.5 μg) or control empty vector (4.5 μg) were introduced into H1299 cells (4.4 × 105 cells/10-cm dish). Where indicated, cells were treated with LLnL (50 μm) 16 h post-transfection. Cells were harvested 21 h post-transfection and analyzed by Western blotting. Experiments were performed in triplicate, and representative images are shown. Levels of p53 were quantified (normalized by β-actin) and the relative p53-72P and -72R levels are shown below the panel. D, pcDNA3-p53-72P or -72R (0.4 μg) together with pCMV-Myc-Mdm2 (3.6 μg) were introduced into H1299 cells (4 × 105 cells/10-cm dish). Cells were pulse-labeled 20 h post-transfection for 30 min and then cultured in chase medium for 1.5 h. Following incubation, cells were harvested at the indicated time points. p53 was immunoprecipitated, and the levels of labeled p53 were detected by autoradiography. Total p53 protein levels were analyzed by Western blotting. Experiments were performed in triplicate, and representative images are shown. Levels of p53 were quantified (normalized by total p53) and the relative p53-72P and -72R levels are shown below the panel. E, immortalized peripheral lymphocytes from healthy donors were subjected to LLnL treatment. Cells derived from 10 homozygotes each for p53-72P and -72R were subjected to analysis. Each sample was run in triplicate and analyzed by Western blotting (supplemental Fig. S3). Quantification was performed using Image J software. Fold accumulation of p53 protein after LLnL treatment was calculated for each sample and shown as a box plot.
Article Snippet: The antibodies used in this study were as follows: anti-p53 goat polyclonal antibody (FL393); anti-p21 rabbit polyclonal antibody (C-19); anti-PIG3 (N-20) and PIG3 (C-20) goat polyclonal antibody; anti-Bax (N-20) mouse monoclonal antibody; anti-c-Myc mouse monoclonal antibody (9E10) and anti-β-actin mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA); penta-His antibody (Qiagen, Valencia, CA); anti-p53 mouse monoclonal antibodies PAb1801 and PAb421 and anti-Mdm2 mouse monoclonal antibody (clone IF-2) (Calbiochem); anti-p53 mouse monoclonal antibody (PAb122) (Monosan, Uden, Netherlands); anti-phospho-p53 (Ser-6, -9, -15, -20, -37, and -46) rabbit polyclonal antibodies and anti-phospho-Smad2 (138D4) Ser-465/467 antibody (Cell Signaling, Beverly, MA); anti-CRP1 antibody (BD Transduction Laboratories); and anti-FLAG mouse monoclonal antibody (M2); and anti-tubulin antibody (clone B-5-1-2) (Sigma).
Techniques: Control, Plasmid Preparation, Western Blot, Transfection, Ubiquitin Proteomics, Expressing, Immunoprecipitation, Labeling, Cell Culture, Incubation, Autoradiography, Derivative Assay, Software
