Mouse monoclonal anti ha clone 16b12

For preparation of total protein parasite extracts, 1 × 10 7 epimastigotes were harvested by centrifugation at 1000 × g and disrupted with the addition of 5 × SDS-PAGE loading buffer (5 × SB). Clarified lysates and insoluble fractions from parasites were prepared as follows: 1 × 10 7 pelleted epimastigotes were resuspended in icecold PBS, disrupted by sonication (four pulses of 20 s each) and centrifuged at 20,000 ×g for 20 min. The fractions obtained (cytoplasmic supernatant and membranous pellet) were mixed separately with 5× SB in order to reach the same final volume for each sample. The different protein extracts were loaded onto 12% SDS-PAGE polyacrylamide gels and subjected to electrophoresis. Gels were transferred to nitrocellulose membranes and proteins detected using mouse monoclonal anti-HA (clone 16B12, Covance), mouse anti-β tubulin (Life Technologies), rabbit polyclonal anti-GFP or rabbit polyclonal anti-TcCyp19 antibodies (the last two kindly provided by Dr. Jaqueline Búa, ANLIS/Malbrán Institutes, Argentina). Horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgGs (Calbiochem) were used as secondary antibodies. All antibodies were used at 1:1000 dilutions in 3% bovine serum albumin in PBS (BSA-PBS) and detected with ECL™ chemiluminescence kit (GE Healthcare), according to the manufacturer's instructions.