Immunoblot Analysis of Parasite Proteins

For immunoblot analysis, parasites were grown in the absence and presence of ATc (1 mg/ml) for 48–50 h. Approximately 3 × 10⁶ freshly released parasites were harvested and resuspended in reducing sample buffer. Equal amounts of protein lysate were separated using NuPAGE 4–12% Bis-Tris gels in MES buffer (Life Technologies) and transferred onto nitrocellulose membranes using the iBlot system (Life Technologies). Membranes were blocked in PBS with 5% skim milk (wt/vol) for 1 h before incubation with one of the primary antibodies (in blocking solution) rabbit anti-HA clone C29F4 (Cell Signaling Technology), 1:4000; mouse anti-cMyc clone 9E10 (Sigma-Aldrich), 1:1000; rabbit anti-Act_239-253 (Angrisano et al., 2012 (link)), 1:4000; and rabbit anti-TgADF (kindly provided by D. Sibley, Washington University, St. Louis), 1:4000. Secondary antibodies used were goat anti-rabbit horseradish peroxidase (HRP) and donkey anti-mouse HRP (Life Technologies) in 1:4000 and 1:2000 dilutions, respectively. Gels were run in duplicate, and membranes were stripped, blocked, and reprobed for multiple analyses.