Figure 1 | Scientific Reports

Figure 1

From: Development of a novel immunoassay to detect interactions with the transactivation domain of p53: application to screening of new drugs

Figure 1

Analysis of the interaction between p53-His and MDM2-GST in cellular extracts. (a) A schematic diagram for detection of recombinant protein p53-His in cells using AlphaLISA. Acceptor beads that are directly conjugated to anti-p53 antibodies capture the TAD of p53. Streptavidin coated donor beads capture the immune complex between the biotinylated anti-His antibodies and His tag, bringing donor beads into close proximity to the acceptor beads. After excitation at 680 nm, acceptor beads receive singlet oxygen from donor beads and produce fluorescent emission at 615 nm. As shown in the lower image, if ligands like MDM2, which can block the TAD domain of p53, exist, acceptor beads are not able to capture p53, resulting in no emission. (b) GST pull-down assays were performed with pENTER-p53 transfected HEK293T cell lysates as prey and MDM2-GST as bait. Immunoblotting was performed using anti-His and anti-GST antibodies. p53-His was pulled down by MDM2-GST but not by GST. When anti-p53 antibodies were added to the cell lysates, p53-His pulled down by MDM2-GST was remarkably reduced. However, isotype IgG had no effect.

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