2.7. Western Blot to Assess Ubiquitylated Proteins

The accumulation of ubiquitylated proteins in parasites treated with 17-AAG (500 nM) for 24 h was evaluated by Western blot analysis. As a positive control, parasites were treated with the proteasome inhibitor MG132 (3 μM) for 24 h. Following treatment, parasites were pelleted by centrifugation for 3 min at 1000× g, and then washed thrice in PBS for medium removal. Protein extraction was then performed for Western blot analysis as described below.

Parasites were lysed with laemmli buffer (2-Mercaptoethanol 0.1%, bromophenol blue 0.0005%, glycerol 10%, SDS 2%, Tris-HCl 63 mM, pH 6.8) at a proportion of 10 μL of buffer for 10⁶ parasites, then boiled for 5 min and cell extracts were stored at −20 °C. Proteins were transferred from a 12% polyacrylamide gel, following electrophoresis, to a Hybond-C nitrocellulose membrane (Amersham, GE Healthcare, Little Chalfont, UK). Transfer was carried out by semi-dry blotting using a BioRad Trans-Blot SD Semi-Dry Transfer Cell at 30 volts for 45 min, with membranes and filter papers soaked in transfer buffer (20 mM Tris-HCl, 15 mM glycine, 20% (v/v) methanol, in distilled water). Membranes were subsequently incubated in a blocking solution for 1 h at room temperature or overnight at 4 °C under agitation. After blocking, each membrane was incubated with 1:1000 FK2 anti-ubiquitin antibody (LifeSensors, Malvern, PA, USA) diluted in fresh TBST buffer with 3% milk for 1 h at room temperature. Secondary anti-mouse antibody conjugated with horseradish peroxidase (HRP) (Promega, Madison, WI, USA) was diluted at 1:10,000 in fresh TBST buffer and each membrane was first incubated with an ECL (Enhanced Chemiluminescence) solution (SuperSignal West Pico Chemiluminescent Substrate Kit, Pierce, Rockford, IL, USA), and then exposed on Kodak photographic film. An antibody against elongation factor 1α (EF1α) (Millipore, Germany) was used for loading control. Western blotting experiments were performed three times.