With the intention of further verifying the interaction between NOT9 and NIP-2-Ac, we performed pull-down experiments using an azide-modified analogue (NIP-2-PEG-N3). The peptide was modified with an azidolysine followed by a double PEG-linker on the N-terminus (a double linker was required for efficient enrichment) and immobilized on dibenzocyclooctyne (DBCO)-magnetic beads via copper-free click chemistry followed by incubation with the purified NOT9-NOT1 complex. Satisfactorily, NIP-2-PEG-N3 was able to enrich NOT9 and NOT1, while the negative control NIP-MUT-N3 showed reduced enrichment (Fig. and supplemental Fig. ). To confirm that the NIP-2-Ac peptide was able to inhibit the interaction between the NOT9 and NIP-WT, we performed the pull-down experiment using the azide-modified analogue NIP-WT-N3 as the pull-down probe in the presence of purified NOT9-NOT1 complex. Addition of NIP-2-Ac indeed led to a significant reduction of the interaction between NIP-WT-N3 and NOT9-NOT1 (Fig. and supplemental Fig. ). Next, we repeated the in vitro pull-down experiment by incubating the beads with HeLa cell lysate, and similar enrichment of NOT9 was observed for NIP-2-PEG-N3 confirming it could interact with the target protein in a more complex biological environment (Fig. and supplemental Fig. ). In this experiment the negative control peptide NIP-MUT-N3 did not enrich NOT9 over the background.