Journal:

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

doi:

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

Journal: Molecular Endocrinology

Article Title: Identification and Characterization of Single Strand DNA-Binding Protein That Represses Growth Hormone Receptor Gene Expression

doi: 10.1210/me.11.9.1291

Figure Lengend Snippet: Fig. 6. Effects of Mutations in the DNA-Binding Site or DOC on the Relative Affinities of the DSBP-DNA Complex To quantitate the effect of mutations in the DNA-binding site by either alterations in the nucleotide sequence or pres- ence of anionic detergent (DOC), EMSA was carried out with equal amounts of liver nuclear extracts and equimolar amounts of the indicated 32P-labeled double-strand DNA probes in the absence of unlabeled specific competitor. After electrophoresis, the dried gels were subjected to analysis using a PhosphorImager, and the amount of bound DNA- protein complex was calculated as a percent of the total amount of probe added per reaction. The data represent mean 6 SEM of three independent experiments. *, P , 0.05 as compared with FP42-DS by one-way ANOVA with Duncan’s Correction for Multiple Comparisons.

Article Snippet: The dried gel was sequentially subjected to autoradiography and analysis via PhosphorImager (Molecular Dynamics).

Techniques: Binding Assay, Sequencing, Labeling, Electrophoresis

Journal: Molecular Endocrinology

Article Title: Identification and Characterization of Single Strand DNA-Binding Protein That Represses Growth Hormone Receptor Gene Expression

doi: 10.1210/me.11.9.1291

Figure Lengend Snippet: Fig. 7. Determination of Dissociation Rates (Half-Life) of the Cognate DNA-Binding Protein for FP42-DS, M2FP42-DS, and M3FP42-DS Panel A, 32P-labeled FP42-DS (lanes 1–5) or M2FP42-DS (lanes 6–10) was incubated with nuclear extracts prepared from liver of adult female mice. After the reactions had reached equi- librium (20 min), a 400-fold excess of homologous unlabeled double-stranded oligonucleotide was added, and aliquots of the mixture were loaded onto a running gel at the indicated time points. After electrophoresis, the dried gels were subjected to analysis using a PhosphorImager. Panel B, Data (mean 6 SEM; n 5 3) from the PhosphorImager analysis of gels similar to that shown in panel A are plotted as the log of the percent bound probe relative to probe bound at the time of addition of the unlabeled competitor (time 0), as a function of time. The half-life of the complexes were derived from the slope of the curves (10); FP42-DS(t 1⁄2) 5 25 min, M2FP42-DS(t 1⁄2) 5 14 min, and M3FP42- DS(t 1⁄2) 5 10 min.

Article Snippet: The dried gel was sequentially subjected to autoradiography and analysis via PhosphorImager (Molecular Dynamics).

Techniques: Binding Assay, Labeling, Incubation, Electrophoresis, Derivative Assay

Journal: Molecular Endocrinology

Article Title: Identification and Characterization of Single Strand DNA-Binding Protein That Represses Growth Hormone Receptor Gene Expression

doi: 10.1210/me.11.9.1291

Figure Lengend Snippet: Fig. 8. Effect of DOC on Dissociation Rate of the Cognate DNA-Binding Protein for FP42-DS In the presence of the indicated concentration of DOC, 32P-labeled FP42-DS was incubated with nuclear extracts prepared from liver of adult female mice. After the reactions had reached equilibrium (20 min), a 400-fold excess of unla- beled FP42-DS was added, and aliquots of the mixture were loaded onto a running gel at the indicated time points. After electrophoresis, the dried gels were subjected to analysis using a PhosphorImager, and the data (mean 6 SEM; n 5 3) plotted as the log of the percent bound probe relative to probe bound at the time of addition of the unlabeled com- petitor (time 0), as a function of time. The half-life of the complexes was derived from the slope of the curves (10); t1⁄2 (min) 5 25 (0% DOC), 21 (0.09% DOC) and 14 (0.15% DOC).

Article Snippet: The dried gel was sequentially subjected to autoradiography and analysis via PhosphorImager (Molecular Dynamics).

Techniques: Binding Assay, Concentration Assay, Labeling, Incubation, Electrophoresis, Derivative Assay