C57BL6 male mice were placed in the open field for 30 min prior to injection of vehicle (saline) or 5 mg/kg i.p. U50488 (Tocris). Two hours later, mice were transcardially perfused with 4% paraformaldehyde (PFA) and 0.1 M PBS. Brains were postfixed overnight in PBS with 4% PFA and then transferred to 20% sucrose in PBS with 4% PFA.

Flasks of T. brucei were seeded at 1 x 104 cells/ml and incubated in the presence of the U50,488 for 3 days. Flasks were set up in triplicate for each inhibitor at multiples of EC50 along with 3 control flasks with no inhibitor added. Pentamidine and U50,488 were added in DMSO in a volume of 0.1% of the culture. Eflornithine was dissolved directly in HMI9-T medium and sterilised by 0.22 mMfiltration before diluting into the cell culture.