Arginine transport inhibitors pentamidine and canavanine down-regulate ADR by the rapid degradation of LdAAP3 and LdPT.

A and B. The effect of arginine transport inhibitors on ADR was checked. Axenic promastigotes were cultured in an M199 medium, and the cells were washed with Earl’s salt solution followed by starvation for two hours in arginine deprived M199 medium. After two hours, arginine transport inhibitors (100 μM pentamidine, 500 μM canavanine and 1 mM NMLAA) were added to the arginine deprived cells, and the cells were further incubated for the following time periods (30 min and 60 min for pentamidine; 60 min respectively for canavanine and NMLAA). The total RNA was isolated from undeprived and arginine deprived cells. The effect of transport inhibitors on ADR was checked by determining the expression levels of LdAAP3 (A) and LdPT (B) using gene-specific probes as described in Materials and Methods. The abundance of the expression of LdAAP3 or LdPT normalized relative to the rRNA of each condition was calculated using ImageJ and is illustrated in the graphs below the gels. The image is representative of three independent experiments. The two asterisks (**) in the figures refer to the two transcripts (5-and 7-kb) synthesized from the LdBPK_310910.1 gene.