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Blood, Vol. 114, Issue 1, 165-173, July 2, 2009
GATA-1 associates with and inhibits p53
Blood Trainor et al.
114: 165
Supplemental materials for: Trainor et al
Isothermal Titration Calorimetry binding experiments
ITC titrations were performed essentially as described40 in 20 mM phosphate buffer pH 6.4 at 25°C. The protein concentrations were determined from A280. In all experiments, the GATA-1 fragment was in the stirred cell and increasing concentrations of p53 fragments were added incrementally. All injections fit the single-binding site mechanism with 1:1 stoichiometry.
Files in this Data Supplement:
- Figure S1. Preliminary studies indicate that erythropoietin (EPO) may repress p53Luc activity by activating or stabilizing GATA-1 (JPG, 95.3 KB)
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K562 cells (2 × 106) were plated in 60 mm dishes and transfected with 150 ηg of CMV renilla luciferase and 0.5 µg of p53Luc; 0.25 µg of CMV p53 was added to two of the samples (bars 2 and 4). Effectene from Qiagen was used as the transfection reagent according to the manufacturers instructions with 8 µl of enhancer and 25 µl of effectene per sample. 7.4 ηg/ml of EPO (Sigma) was added to one set of samples (bars 3 and 4). After 24 hours the dual luciferase assay was performed and firefly luciferase activity was normalized to renilla luciferase. The firefly luciferase activity from sample 2 was set to 100% and all other values normalized to that. Results are the means from six experiments and error bars represent the standard deviation. The means of samples 2 and 4 were compared by Student’s t test and results were not statistically significant. A 10% increase in GATA-1 mRNA level in response to EPO was measured by QPCR, but the increase in GATA-1 mRNA levels was also not statistically significant.
- Figure S2. Amino Acids 232 to 256 of the linker between the two zinc fingers of GATA-1 are required for inhibition of p53 (JPG, 66.8 KB)
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(A) A Schematic of the N-finger and linker of GATA-1 (amino acids 196 to 256) that was removed to generate ΔNFL (top, Fig. 5A) and of the region of the N-finger that was returned to ΔNFL to generate ΔNL (middle) and ΔNL2 (bottom) is shown. (B) A transfection into 6C2 cells was performed as above. CMV renilla luciferase, p53 Luc reporter plasmid, and CMV p53 were present in all samples and expression plasmids for GATA-1 and mutants were added as indicated (1, 2, and 3 µg). A sample in which empty vector was added was set at 100% and all values were normalized accordingly. Values are averages of six experiments and the error bars indicate the standard deviations. Means of the two highest DNA concentrations of all mutants were compared to that of equal weights of WT and ** p < 0.005, *** p < 0.0005. (C) CMV renilla luciferase and 1 µg FR7Luc, a GATA-1 responsive reporter plasmid, were tranfected into QT6 fibroblasts with increasing amounts of mouse GATA-1, ΔNL, ΔNL2 and ΔCF expression plasmids as indicated (0.5, 1, or 2 µg). Normalization and error bars are as in (B) and values are the average of 3 experiments. The differences between the means of WT vs. ΔNL and ΔNL2 are not statistically significant by Student’s t test.
- Figure S3. GATA-1 ZFD and GATA-1 L + CF bind p53TAD with the same affinity (JPG, 236 KB)
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(A) A typical ITC thermagram obtained by successive additions of p53TAD to GATA-1 L + CF. (B) A typical ITC thermagram obtained by successive additions of p53TAD1 to GATA-1 L + CF. (C) Dissociation constants (Kd) for the binding of GATA-1 fragments to p53 fragments as determined by ITC. The ITC curves were fit using a single-binding site to solve for the stoichiometry (N), the dissociation constant (Kd) the change in enthalpy (ΔH). The experimental conditions are as described in the experimental procedures. All injections fit the single binding site mechanism with 1:1 stoichiometry.
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