Immunoprecipitation and Western Blotting of Parasite Proteins

For Western blotting analysis, the samples were lysed in 300 μL IP buffer and incubated with 30 μL Pierce anti-HA magnetic beads (Thermo) overnight at 4°C, following which the beads were washed three times with IP buffer and the bound proteins eluted with 30 μL 3x Sample Loading Buffer (NEB) at 95°C for 10 min. 5% of the post-IP lysate supernatant and 15% of the immunoprecipitate were separated by SDS-PAGE and transferred to a nitrocellulose membrane as above. The membrane was blocked with 2% w/v skim milk powder, 0.1% v/v Tween 20 in PBS for 1 h at room temperature, then incubated with primary antibodies in blocking buffer overnight at 4°C. Primary antibodies used were: 1:1000 rat anti-HA (Roche #11867423001), 1:1000 mouse anti-ROP1 (Abnova #MAB17504), 1:1000 rabbit anti-C1QBP (Abcam #ab270032), 1:10,000 rabbit anti-GAPDH (Proteintech #10494-1-AP), and 1:1000 rabbit anti-GRA29 [15 (link)]. Blots were stained with secondary antibodies for 1 h at room temperature: 1:10,000 goat anti-mouse IRDye 680LT (Li-Cor #925–68020), 1:10,000 goat anti-rat IRDye 800CW (Li-Cor #925–32219), 1:10,000 donkey anti-rabbit IRDye 680LT (Li-Cor #925–68023), and 1:10,000 donkey anti-rabbit IRDye 800CW (Li-Cor #925–32213). Blots were visualised using an Odyssey CLx scanner (Li-Cor).