FIG. 6.

The L. donovani GFPK7 growth defect is linked to limited de novo protein biosynthesis. (A) Metabolic labeling. (Left) Control and LdGFPK7 promastigotes were inoculated into amastigote medium, and the number of parasites was determined microscopically by cell counting. (Right) De novo protein biosynthesis was assessed at the time points indicated by metabolic labeling with Pro-Mix containing l-[³⁵S]methionine and l-[³⁵S]cysteine (GE Healthcare). Protein amounts were normalized by equal cell numbers. An unpaired t test was applied to the 24- and 48-h time points. **, P value of 0.0018; ***, P value of 0.0001. (B) Western blotting. Total protein extracts or purified phosphoproteins obtained from wild-type (WT) or GFPK7 (7) parasites 12 h and 24 h after the induction of amastigote differentiation were separated by SDS-PAGE, and the abundance of eIF2α was revealed by Western blotting. The levels of α-tubulin expression were assessed for normalization purposes. Molecular mass standards are indicated (in kilodaltons).