Pom binds to TP53RK. (A) A 3′-n-butylamine derivative of Pom was synthesized and attached via linker to generate Pom-based affinity reagent. (B) MM.1S whole cell lysates were incubated with Pom-beads in the presence or absence of competitor (1 mM free Pom) for 1 hour. After elution, samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. DMSO, dimethyl sulfoxide. Specific bands pulled down by Pom-beads were subjected to mass spectometry analysis. Protein lysates from MM.1S pulled down by Pom-beads (C), Thal-beads (D), or whole cell lysate (W.L.) were subjected to western blotting using anti-CRBN Abs (top panel). Membranes were subsequently blotted with anti-TP53RK Abs without stripping. IB, immunoblot. (E) Binding of Pom and recombinant TP53RK was assessed by saturation-transfer difference NMR spectrometry. (F) ITC analysis of TP53RK/TPRKB complex and Pom. (G) DSF measurements showed that Pom, but not Len, stabilized the complex. ADP, adenosine 5′-diphosphate. (H) Recombinant C-terminal Myc-DDK-tagged TP53RK and TPRKB proteins from HEK-293FT cells were subjected to in vitro autophosphorylation assay.