Loss of CSAR1 reduces the amount of soluble tubulin available for polymerization. (A) Western blots quantified in Fig. 6 A. Toxoplasma polymerized tubulin structures are stable in detergent such as Triton-X-100 and can be separated from unpolymerized tubulin by centrifugation (see Materials and methods). Equal volumes of soluble versus insoluble, assembled cytoskeleton from four biological replicates (1–4) with four technical replicates each (A–D) of either Parental (Par.) or Δcsar1 parasites were separated by SDS-PAGE and quantified by Western blot against Toxoplasma β-Tub. (B) Comparison the sensitivity of parental (ERK7^AID) and Δcsar1 parasites to the tubulin-polymerization inhibitor orazylin. Parasites were grown for 10 d in the indicated concentrations of oryzalin, and the numbers of resulting plaques were normalized to the vehicle control. Significance was calculated by one-way ANOVA followed by Tukey’s multiple comparison test (n.s., not significant). (C) To validate the CSAR1 RING mutant, we amplified the 5′ end of the gene from the genomic DNA, using primers that annealed in the indicated regions. Note that the 5′ primer anneals in the csar1 locus outside of the targeting region of the construct used, to ensure that the amplicon results from the endogenous locus. 5′ primer (5′-ACA​TCG​GCG​GAG​GAA​GAG​AG-3′), 3′ primer (5′-GTA​GAC​TTC​TCC​CTT​CAG​CGG​C-3′). The resulting amplicon was purified and then sequenced with the same primers. Sequencing confirmed correct integration into the endogenous locus and wild-type sequence outside of the area targeted for mutagenesis. A region of the resulting chromatogram is shown with the three mutated residues shown in red. Source data are available for this figure: SourceData FS5.