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Fig. 8 | BMC Biology

Fig. 8

From: A yeast two-hybrid system for the screening and characterization of small-molecule inhibitors of protein–protein interactions identifies a novel putative Mdm2-binding site in p53

Fig. 8

The conserved Segment 1 in p53’s DNA-binding domain promotes interaction with Mdm2. a Alignment of amino acid residues 100–143 of Homo sapiens p53 with the homologous amino acid sequences of Homo sapiens p63, Homo sapiens p73, Mus musculus p53, Gallus gallus p53, Drosophila melanogaster p53, and Caenorhabditis elegans p53. Conserved Segments 1, 2, and 3 are indicated in yellow, cyan, and red, respectively. Location of the conserved Box II is indicated by the black rectangle. b Overnight cultures of ABC3Δ cells containing plasmids encoding either Gal4 AD-Csm1/Gal4 BD-Dsn1, Gal4 AD-p53 (1–52)/Gal4 BD-Mdm2, Gal4 AD-p53 (1–143)/Gal4 BD-Mdm2, Gal4 AD-p53 (1–143-Seg1A)/Gal4 BD-Mdm2, Gal4 AD-p53 (1–143-Seg2A)/Gal4 BD-Mdm2, or Gal4 AD-p53 (1–143-Seg3A)/Gal4 BD-Mdm2 in non-selective medium were washed in water and inoculated at OD600 = 0.2 into selective medium containing DMSO or nutlin at the indicated concentrations. Growth of the cultures was monitored by recording the absorbance at 600 nm after 0, 23, 30, 47, and 54 h following inoculation. Ends of the vertical bar indicate the OD600 values of the duplicate cultures. c Mdm2-HA, full-length p53 (p53-FL), full-length p53 with Seg1A mutation (p53-FL-Seg1A), p53 (1–116), p53 (1–143), and p53 (1–143-Seg1A) were synthesized by in vitro translation. The binding assay was performed by incubating Mdm2-HA with the different p53 variants, followed by immunoprecipitation of Mdm2-HA using an anti-HA antibody and measuring the amount of co-immunoprecipitated p53 by western blotting analysis using anti-p53 (DO-1) antibody. Input (IN), immunoprecipitated proteins (IP), and eluate from control IP performed without Mdm2-HA (CON) were loaded on SDS-PAGE gels

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