Fig 2: Immunoprecipitation–mass spectrometry identifies potential serum ligands of FHR proteins. (a) Schematic overview of the immunoprecipitation-MS workflow. Recombinant His-tagged FHR proteins were immobilized on Ni–NTA beads and incubated with pooled human serum. After extensive washing, bead-bound proteins were subjected to on-bead reduction, alkylation, and tryptic digestion prior to LC–MS/MS analysis. (b) Combined interaction network of proteins identified in the FHR-mediated immunoprecipitation (note proteins are indicated here by their gene nomenclature). Gray nodes represent FHR family proteins (bait proteins included in the assay), and magenta nodes indicate secreted proteins. Candidate ligands in bold (i.e. CTSG, C4, PPBP, and MBL2) were chosen for further validation.

Fig 3: Solid-phase binding assays validate interactions between recombinant FHR proteins and selected serum ligands. When testing C4 and CTSG binding, C4 and CTSG were immobilized on microtiter plates and incubated with the FHR proteins. Whereas testing MBL2, and PPBP binding with FHR proteins, recombinant FHR proteins, FHL-1, and FH were immobilized on microtiter plates and incubated with the indicated candidate ligands: (a) Complement C4 (C4), (b) cathepsin G (CTSG), (c) mannose-binding lectin 2 (MBL2), and (d) platelet basic protein (PPBP). Bound ligands were detected using ligand-specific antibodies followed by HRP-conjugated secondary antibodies and colorimetric detection at 450 nm. Bar graphs represent mean ± SEM from three independent experiments, each with four technical replicates (n = 3).