*2.14. Western Blotting*

For western blotting detection, epimastigotes were lysed with 1 mL RIPA buffer supplemented with 1 mM phenylmethanesulfonyl fluoride and 5 mM leupeptin, for 30 min at 4 ◦C. Then, homogenates were centrifuged at 16,000 rpm for 10 min. Aliquots of the supernatants (containing 100 μg total protein) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA), which were blocked with 5% milk in PBS plus 0.1% (*w*/*v*) Tween 20, probed overnight at 4 ◦C with the primary mouse anti-GAPDH antibody (1:500, Sigma-Aldrich), and detected using horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA). The loading control was probed with a primary rabbit anti-tubulin antibody (1:500, Sigma-Aldrich) and detected using an HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000, Santa Cruz Biotechnology). Luminescence was detected using an ImageQuant LAS 4000 digital imaging system (GE Healthcare Life Sciences, Amersham, UK) after the reaction with LuminataTM Forte Western HRP Substrate (Millipore, Billerica, MA, USA). Densitometric analysis was performed using ImageJ software version 1.50i (NIH Image, Bethesda, MD, USA) with background correction.
