Peptide-binding assays

Peptide-binding ELISA was carried out in a white 96-well microtiter plate (Costar), which was coated with 1 μg of ubiquitin or 1 μg of NEDD8 (Bio-techne R&D systems) and incubated overnight at 4°C. The plate was blocked using BSA 3% (Sigma) for 1 h at RT. From 0-100 ng of biotinylated peptide-37 (that is H-FIPAQLHFHWRSGSG(LysBiotin) -NH2, in the format Amine-PEPTIDE-GSG-Lys(Biotin)-amide) was added and incubated for 1 h at 37°C (Figure 2C). In another set of experiments, 100 ng of peptides was incubated for 1, 5, 20, and 60min at 37°C (Figure 2D). Binding was detected using streptavidin-HRP-conjugated protein coupled to chemiluminescence on Varioskan Lux Reader (Thermo Fisher Scientific).

Identification of peptides with bioactivity in ubiquitin assays. (A) Ubiquitin assays driven by the E3 ubiquitin ligase MDM2 were assembled with individual peptides from the reactive pools derived from the 60 peptides in Supplementary Table 3. Representative assay showing that specific peptides such as 12 and 37 reproducibly inhibited MDM2 catalyzed ubiquitination (from the left, lanes 2 and 4 vs. lane 1 DMSO-only control). (B) Ubiquitin assays driven by the E3 ubiquitin ligase CHIP were assembled with individual peptides (labeled Φ) that were active in inhibiting MDM2-driven reactions (12 and 37) and a non-active ubiquitin-binding peptide 44 (Figure 2B). D represents DMSO-only control. Reactions were processed by immunoblotting to detect changes in p53 ubiquitination as indicated in the methods. (C,D) Analysis by ELISA of peptide-37 activity with a C-terminal biotin tag. Ubiquitin or NEDD8 was coated on the solid-phase to mimic the original peptide-phage screen. Peptides were added and then either titrated in duplicates as indicated from 6.25–100 ng for 60 min (C) or else fixed peptide levels (100 ng) were added in duplicates as a time course from 1 to 60 min (D). The data are tabulated as binding in ECL units.