Figure 1. Biochemical characterization of the ACBD3:PI4KB complex.

(A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments. ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD)¹⁶. PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes¹. (B) In vitro pull-down assay. Pull-down assays were performed using NiNTA-immobilized N-terminal His₆GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue. The asterisks mark the bands corresponding to specific interactions. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation. AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. SPR analysis of the PI4KB binding to immobilized ACBD3. Sensorgrams for four concentrations of PI4KB are shown.