Do ASPP1 and ASPP2 bind directly to p53 family of proteins?
Having established that the proteins of interest are monomers in solution and folded at 20°C, we tested if there is direct interaction between the ASPP proteins and the corresponding DNA-binding domains of the p53 family of proteins using ITC and analytical gel filtration. Different ratios were tested to establish the strength of the interactions. The interaction between ASPP2 and p53 DBD was used as a control since it has been reported that the two proteins interact (16,17,19). Our ITC experiments provide for the first time quantitative data and show that both ASPP1 (Figure 3A–C) and ASPP2 (Figure 3D–F) interact directly with all three p53 family of proteins, namely p53, p63 and p73. The Kd values for all the interactions are similar and are in the lower micro-molar range (between 0.5 and 5 µM) (Table 1). These values are in excellent agreement with the recently published Kd value of binding for p53 DBD to 53BP2 (namely ASPP2), with virtually identical constructs (19). ITC provides an
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