Pf3D7_0811400 interaction with Ub-Dha probe and PfRbx1.

A) Schematic of Pf3D7_0811400. Domains and truncations used to generate protein for labelling and interaction studies are shown. Five predicted N-terminal α-helices (amino acids 1–88) were truncated to investigate their role in protein-protein interactions. Depicted domains include: FLAG (gray dotted), coils (wide stripes), 17 amino acid linker (white), DUF2009 (narrow stripes). B) Validation of Pf3D7_0811400 interaction with Ub-Dha probe. HEK293T cells were transfected with either an empty vector, FLAG-tagged Pf3D7_0811400, or FLAG-tagged Pf3D7_0811400ΔN. Whole cell lysates were labelled with biotin-Ub-Dha in the presence of ATP and additional E1 enzyme. The proteins were then co-immunopurified on anti-FLAG resin. Proteins were visualised by immunoblot with HRP-conjugated streptavidin (top panel) or Anti-FLAG (bottom panel) to confirm a ~10 kDa shift in protein size in the presence of the probe. C) Pf3D7_0811400 interaction with PfRbx1 via its N-terminal domain. Myc-tagged PfRbx1 was co-expressed with either FLAG-tagged Pf3D7_0811400 WT or the ΔN truncated mutant in HEK293T cells. FLAG-tagged PfFBXO9 in the presence of PfSkp1 was used as a positive control, while an empty vector (EV) in the presence of both PfRbx1 and PfSkp1 served as a negative control. Immunopurification using anti-FLAG beads was performed on all samples and proteins were visualised by anti-myc or anti-FLAG. WCE=whole cell extract. Images of full membranes are included in S6 Fig.