Localization of the Cyclopropane fatty acid synthase (CFAS) protein is affected by the downstream flanking sequence (FR) of CFAS in Leishmania mexicana log phase promastigotes. Log phase promastigotes of cfas−/+HA-CFAS-3’FR (A) and cfas−/+HA-CFAS (B) were permeabilized with ethanol and processed for immunofluorescence microscopy. HA, immuno-staining with a mouse anti-haemagglutinin antibody (1:800), followed by a goat anti-mouse IgG-FITC antiserum (1:1000); ER, immuno-staining with a rabbit anti-Trypanosoma brucei Bip antibody, followed by a goat anti-rabbit IgG-Texas Red antiserum; Merge, overlay of HA- and ER-stainings; DNA, Hoechst 33242 staining; DIC, differential interference contrast images. Scale bars =10 µm. (C) Percentages of co-localization between HA-staining and ER-staining were quantified in cfas−/+HA-CFAS-3’FR and cfas−/+HA-CFAS parasites (50 randomly selected cells per group were analyzed) using the Image J JACoP program.