Immunoprecipitation of Ts-Pmy and C9 Interaction

To further verify the protein interaction between the human complement C9 and the native Ts-Pmy, immunoprecipitation was performed with T. spiralis adult extracts as described previously [18 (link)]. Protein G MicroBeads (Miltenyi Biotec, Germany) were pre-incubated with 3 μg of human C9 (Merck, Germany) and 2 μg of anti-C9 mAb (IgG1, Abnova, Taiwan) for 30 min on ice. In total, 40 μg of T. spiralis adult extracts was then added and incubated overnight at 4°C with rotation. Beads were washed four times with washing buffers (1% NP40, 50 mM Tris buffer, pH 8.0) before being added with pre-heated 1× SDS gel loading buffer to elute proteins. Samples were separated by SDS-PAGE and probed with anti-Ts-Pmy mAb (7E2) [19 (link)]. IRDye 800CW-labeled goat anti-mouse IgG (LI-COR, Germany) was used as the secondary antibody.