Journal:

Article Title: DNA damage activates p53 through a phosphorylation-acetylation cascade

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Figure Lengend Snippet: Acetylation of p53 and p53 fragments by p300 and PCAF. Wild-type human p53 or truncated p53 fragments were acetylated with either PCAF or p300 at 37°C for 20 min as described in Materials and Methods, and the reaction products were analyzed by SDS-PAGE. P300 acetylation (14C-Label) is depicted in the radioactive image in C; the corresponding Coomassie brilliant blue-stained image (CBB) is in A. PCAF acetylation is in D; the corresponding Coomassie brilliant blue-stained image is in B. Histone H1 served as a positive control for acetylation (Herrera et al. 1997). (Lanes M) Molecular weight markers; (lanes 1) full-length wild-type, baculovirus-produced human p53; (lanes 2) p53(1–355); (lanes 3) p53(283–393); (lanes 4) p53(318–393); (lanes 5) histone H1.

Article Snippet: The incorporation of [ 14 C]acetate into p53 was measured by use of PhosphorImager analysis of the Enhance-impregnated (Dupont) SDS–polyacrylamide gels by use of Imagequant software (Molecular Dynamics, Inc.).

Techniques: SDS Page, Staining, Positive Control, Molecular Weight, Produced

Journal:

Article Title: DNA damage activates p53 through a phosphorylation-acetylation cascade

doi:

Figure Lengend Snippet: Activation of sequence-specific binding by acetylation of p53 with p300 and PCAF. Baculovirus-produced wild-type p53 was acetylated with p300 or with PCAF as described in Materials and Methods, and the reaction products then were used in electrophoretic mobility shift assays as described by Anderson et al. (1997). (A) Radioactive images of the EMSA gels; the ingredients present during the p53 modification reactions are indicated at top. (Lanes 14,15) The order of p300 and PCAF additions are indicated by superscripts; (lane 7,11) unacetylated CoA was added in place of acetyl–CoA (Ac–CoA). The p53-shifted radioactive probe appears as a band near the top of the gel; free probe is at the bottom. (B) Parallel acetylation reactions were performed with 14C-labeled acetyl–CoA, and the reactions were fractionated by SDS-PAGE. Shown is the radioactive image of the gel. (Lane 1) Reaction with p53 and p300, (lane 2) reaction with p53 and PCAF; (lane 3) reaction with p53 incubated with PCAF and then also with p300; (lane 4) reaction with p53 incubated with p300 and then with PCAF.

Article Snippet: The incorporation of [ 14 C]acetate into p53 was measured by use of PhosphorImager analysis of the Enhance-impregnated (Dupont) SDS–polyacrylamide gels by use of Imagequant software (Molecular Dynamics, Inc.).

Techniques: Activation Assay, Sequencing, Binding Assay, Produced, Electrophoretic Mobility Shift Assay, Modification, Labeling, SDS Page, Incubation