FIGURE 1:

Incorporation of analogue-sensitive mutations into TbPLK in procyclic cells. (A) A schematic of the TbPLK loci and the strategy used to introduce analogue-sensitive mutations. Primers flanking the positions of the C57V (black) and L11G (red) mutations were used to amplify an 811–base pair fragment of the TbPLK gene. If the L11G mutant is present, a unique AvrII restriction site is present in the fragment. (B) PCR using the primers described in A from genomic DNA isolated from cells expressing only Ty1-TbPLK^as, YFP-TbPLK^as, and wild-type cells. The fragment was digested with AvrII to test for incorporation of the analogue-sensitive mutations. The fragment and the product of the AvrII digest for each isolated genomic DNA were run on an agarose gel. Only the fragments isolated from the TbPLK^as cell lines were sensitive to AvrII, showing that the mutations were present. (C) Cell lysates from Ty1-TbPLK^as, YFP-TbPLK^as, and wild-type cells were fractionated by SDS–PAGE, transferred to nitrocellulose, and incubated with an antibody against TbPLK and tubulin as a loading control. (D) Wild-type and TbPLK^as cells were treated with different concentrations of 3MB-PP1 or a vehicle control, and their growth was monitored with a cell counter for 12 h. Error bars, SD of three biological replicates.