Rat anti ha mab 3f10

Parasite samples were centrifuged at 500 g for 5 min at room temperature (RT), and pellets were stored at −20°C until the time of protein extraction. To isolate parasites from RBCs, the pellets were thawed and resuspended in 0.15% saponin for 5 min at RT. Lysed RBCs were removed by washing three times with PBS. Parasite pellets were resuspended in NuPAGE LDS sample buffer (Thermo Fisher) containing 2% β‐mercaptoethanol and incubated at 95°C for 5 min. Proteins were resolved by SDS–PAGE on 4–12% gradient gels and transferred to nitrocellulose membranes. To detect EcDPCK‐mCherry constructs, membranes were blocked in 5% milk and probed overnight at 4°C with 1:10,000 rabbit anti‐mCherry (vide supra). They were then incubated for an hour at RT with 1:10,000 donkey anti‐rabbit HRP‐linked secondary antibodies (GE healthcare, NA934). Protein bands were detected on X‐ray film using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific), according to the manufacturer’s protocol. Membranes were stripped of antibody with 200 mM glycine (pH 2.0) for 5 min and probed with 1:2,500 rat anti‐HA mAb 3F10 (Roche) in order to detect api‐SFG which contains a C‐terminal HA tag (Swift et al,2020b (link)). After incubation with 1:5,000 goat anti‐rat HRP‐linked secondary antibodies (GE healthcare, NA935), proteins were detected as described above.