Fig 1: Increased protein ubiquitination and mitochondrial protease activity in WT parasites treated with NFX. (A) Evaluation of proteins conjugated to ubiquitin in WT parasites non-treated (NT) or treated with NFX (7 μM, 3 days) or grown at 37°C (3 days). Following treatment, parasites were collected by centrifugation and washed in PBS, and the protein extract was obtained as described. Total protein extract (50 μg) was run on SDS-PAGE (12%) and Western blot anti-ubiquitin (Abcam/ab7780) was performed. (B) Protein extracts derived from parasites (1 × 108) grown in the absence or presence of NFX were tested for proteosome-related protease activity. Total protein extracts (300 μg) were incubated at 28°C in buffer Tris–HCl pH 7.4 containing 50 μM of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (50 μM). AMC release was followed fluorometrically at λ ex = 365 nm and λ em = 440 nm. * denotes statistical differences (p < 0.05) compared with WT non-treated parasite extracts (two-tailed unpaired t-test).
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