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. 2016 Jun 1;11(6):e0156718. doi: 10.1371/journal.pone.0156718

Fig 4. Comprehensive screening to identify p53-binding E3 ligases.

Fig 4

(A) AlphaScreen assay to detect the binding between p53 and 258 E3s including eight negative controls. In this experiment, binding of all E3s to biotinylated p53 and biotinylated DHFR was measured, and the relative value was calculated as follows: value from E3 and p53 / value from E3 and DHFR. Green arrowheads indicated the negative controls. (B) In vitro ubiquitination assay using p53-binding E3s obtained in (A). p53-V5 was mixed with each E3, and the ubiquitination assay was carried out using His-ubiquitin. Left panel, p53-V5 was precipitated with anti-p53 antibody and detected with anti-ubiquitin antibody. Right panel, His-ubiquitin was pull-down with Ni-sepharose, and ubiquitinated p53 was detected with anti-p53 antibody.