Summary of hit E3 ubiquitin ligases (E3s).

<p>E3s for which the relative luminescence signals in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156718#pone.0156718.g004" target="_blank">Fig 4A</a> were higher than 8 are listed. E3s indicated in bold character have been previously reported to interact with p53.</p

Development of AlphaScreen assay.

<p>(A) AlphaScreen assay to detect binding between p53 and MDM2. Crude translation mixtures of biotinylated p53 (WT or ∆I) and FLAG-MDM2 (0.75 μL each) were used for the assay. FLAG-DHFR was used as a control for MDM2. (B) A reaction mixture containing p53 and MDM2 prepared under the same conditions as those in (A) was mixed with 2, 10, or 50 μM Nutlin-3. Biotinylated FLAG-peptide was used as a control to measure the interference of Nutlin-3 in the AlphaScreen assay. (C) Validation of the quality of the AlphaScreen assay. Z′ factor was calculated from the reaction of wild-type p53 and MDM2 (positive control, n = 40, square) and the reaction of p53 ∆I mutant and MDM2 (negative control, n = 40, circle). In (A) and (B), all data are expressed as mean values of three independent experiments with error bars indicating standard deviations.</p

Model assay using p53 and MDM2.

<p>(A) Expression of recombinant proteins of p53 and MDM2 with the wheat cell-free system. Wild-type (WT) and mutant p53 lacking the amino acid residues required for binding with MDM2 (∆I) were synthesized as N-terminal biotinylated form. Wild-type MDM2 and its mutant with a single amino acid substitution in the catalytic core cysteine residue (C/S) were synthesized as N-terminal FLAG-tagged protein. Biotinylated and FLAG-dihydrofolate reductase (FLAG-DHFR) were used as negative controls for p53 and MDM2, respectively. The total translation mixture of each protein was centrifuged, and the whole translation mixture (W) and supernatant (S) were subjected to SDS-PAGE followed by immunoblot analysis (IB) using the antibodies indicated below. (B) Binding assay of biotinylated p53 and FLAG-MDM2 using a conventional immunoprecipitation (IP) assay. Fifteen microliters of crude biotinylated p53 (WT or ∆I) and FLAG-MDM2 were mixed and precipitated with anti-FLAG-antibody. Immunopreciptates were detected with anti-FLAG antibody and anti-biotin antibody. FLAG-DHFR was used as controls for FLAG-MDM2. (C) <i>In vitro</i> ubiquitination assay using biotinylated p53 and FLAG-MDM2. The crude translation mixtures of biotinylated p53 (WT or ∆I) and FLAG-MDM2 (WT or C/S) were mixed and then the reaction mixture containing ATP and HA-tagged ubiquitin (HA-Ubi) with or without UbcH5b was added. After the ubiquitination reaction, biotinylated p53 was pulled down with streptavidin (STA) magnet beads and subjected to SDS-PAGE followed by immunoblot analysis using anti-HA-antibody (for ubiquitin) and anti-biotin antibody (for p53).</p

Study overview.

<p>cDNA clones encoding the E3 ubiquitin ligases (E3s) used in this study were divided into two categories. The clones in category B could be used directly for split-primer PCR, but those in category A were inserted into vectors that cannot be used directly for split-primer PCR and thus were transferred into the pDONR221 vector by using the Gateway system. The names of the vectors and the origins of the clones are indicated.</p

RNF6-dependent ubiquitination and degradation of p53 in cells.

<p>(A) Effect of RNF6 on p53 expression level. H1299 cells were co-transfected with a fixed amount of p53-V5 plasmid (25 ng) and an increased amount of FLAG-RNF6 plasmid (100 to 400 ng). Cell lysates from each reaction were subjected to SDS-PAGE followed by immunoblot analysis using anti-V5 antibody and anti-FLAG antibody. The band intensity of p53 in each reaction was quantified using imageJ software. Relative intensities normalized with the reaction of empty vector were indicated below the blot detected with anti-V5 antibody. (B) Co-transfection of p53-V5 with the wild-type (WT) or activity-deficient mutant (C/S) RNF6. (C) Stability of p53 in the presence of wild-type or C/S mutant of RNF6 was investigated by a pulse-chase assay. H1299 cells co-transfected with p53-V5 and wild-type or C/S mutant of FLAG-RNF6 were treated with 100 μg/ml of cycloheximide (CHX) for indicated time periods. The amount of p53-V5 detected by immunoblot analysis was quantified as same procedure as (A), and relative intensities normalized with the reaction without cycloheximide were indicated below the blot. (D) Endogenous RNF6 in H1299 cells were knockdown by siRNAs targeting RNF6, and overexpressed p53 was detected by immunoblot analysis (upper). The intensity of p53 band in each lane was quantified and intensites relative to the control were indicated below. Efficiency of RNF6 knockdown was confirmed with reverse transcription-quantitative PCR (lower).</p

Comprehensive screening to identify p53-binding E3 ligases.

<p>(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) <i>In vitro</i> 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.</p