The ability of NIP-2 to inhibit RNA binding by NOT9 suggested it could inhibit bulk deadenylation by the CCR4-NOT complex. To evaluate this, we expressed and purified a CCR4-NOTcore complex which contains NOT9, the deadenylases CCR4 and CAF1, as well as the NOT1 section (residues 1093-1607) that connects these subunits (Fig. ). We expected this complex to be suitable for the evaluation of NOT9 inhibitors as it was previously shown to be able to have increased deadenylation efficiency for poly(A) containing RNAs in comparison with CAF1/CCR4 alone. The reconstitution of the CCR4-NOTcore complex was achieved according to previously reported protocols where two individual protein complexes (CCR4-CAF1 and NOT9-NOT1) were co-expressed and purified separately. These complexes were then combined and purified to reconstitute the CCR4-NOTcore complex and its formation was confirmed by mass photometry (supplemental Fig. ). With the CCR4-NOTcore complex in hand, a gel-based deadenylation inhibition experiment was conducted where 100 nM FAM-labelled A20 RNA was incubated with a fixed concentration of core complex and increasing concentrations of NIP-2-Ac. After incubation, the samples were separated on a TBE-Urea gel to visualize the integrity of the A20 RNA. Dose-dependent inhibition was observed for NIP-2-Ac and the deadenylation process was significantly halted at a 10 μM concentration (Fig. , supplemental Fig. /18/19). As a negative control, a similar inhibition experiment was performed with the mutant peptide NIP-MUT-Ac which was not able to block RNA degradation even at high peptide concentrations (Fig. , supplemental Fig. ). Next, an in vitro deadenylation experiment was conducted in the presence of only the CCR4-CAF1 exonuclease complex. In this case, a much lower degree of degradation was observed even at a double protein concentration and double incubation time in comparison to CCR4-NOTcore confirming the role of NOT9 in increasing the deadenylation rate. However, no inhibition was found at a 10 μM concentration of NIP-2-Ac (Fig. , supplemental Fig. ) confirming that NIP-2's capacity for inhibition of RNA deadenylation occurs via NOT9.