Figure 4.

Phosphoproteomic investigation of CDPK3 and SCE1 egress. A, overview of the strategy for phosphoproteomic investigation of CDPK3-regulated egress. Wild-type, Δcdpk3Δsce1, and Δcdpk3 parasites were grown in light, medium, or heavy SILAC labeling medium, respectively. After labeling, parasites were treated with A23187 for 1.5 min to trigger egress-signaling events, parasites were harvested by needle passage, and host cell debris was removed by differential centrifugation. SILAC-labeled samples were mixed 1:1:1 before tryptic digestion. Peptides were fractionated by strong cation exchange (SCX) chromatography, phosphopeptides were enriched on TiO₂ beads, and the resulting samples were analyzed by MS/MS. B, the number of high-confidence Toxoplasma and human proteins, peptides, and phosphopeptides identified following MaxQuant analysis. C, histograms of median-centered log₂ SILAC ratios, Δcdpk3Δsce1/wild type (M/L), Δcdpk3/wild type (H/L), and Δcdpk3/Δcdpk3Δsce1 (H/M). D, the number of high-confidence phosphosites that are significantly up- or down-regulated in Δcdpk3 and Δcdpk3Δsce1 (relative to wild-type parasites) and restored to near wild-type levels in Δcdpk3Δsce1 (relative to Δcdpk3).