Figure 1. 2-DE display of protein phosphorylation events following in vivo stimulation of Ca²⁺ pathways in Toxoplasma gondii tachyzoites.

Toxoplasma parasites were incubated in media containing ³²[P]-sodium orthophosphate to radioactively label intracellular ATP pools and calcium-dependent signal transduction was stimulated in vivo by treating free tachyzoites with Ca²⁺-mobilizing drugs, as detailed in Materials & Methods. As negative controls parasites were pre-incubated in the presence of membrane-permeable Ca²⁺-chelator BAPTA-AM (A) or treated with DMSO solvent alone (C), and for Ca²⁺ pathway stimulation parasites were incubated with 1% (ie. 172 mM) ethanol (B) or 100 µM ionomycin (D), respectively. Arrows indicate the positions of 50 ³²[P]-labeled 2D protein spots that are exclusively phosphorylated following Ca²⁺ signaling pathway stimulation. E) A false colour image overlay of corresponding unstimulated (C, green) or stimulated (D, red) autoradiographs showing the relative position of multiple confident LC-MS/MS protein identifications (<1% false protein identification rate). A detailed list of proteins IDs to accompany this figure is provided (Supplementary Table S1). F) Radio-immunoprecipitation analyses confirm the phosphorylation of HA-tagged candidate parasite phosphoproteins HA-PKA-R, PP2C-HA, ARM1-HA, MLC1-HA. Free tachyzoites expressing HA-tagged parasite proteins were radioactively labeled with ³²[P]-sodium orthophosphate and treated with 1% ethanol in the presence (+) or absence of BAPTA-AM (-), as indicated. Anti-HA Western blots (αHA, left panels) and corresponding autoradiographs (³²[P], right panels) of radio-immunoprecipitates demonstrate a Ca²⁺-dependent increase in the phosphorylation of HA-reactive tachyzoite protein bands (arrows). Abbreviations: PKA-R, cAMP-dependent protein kinase A regulatory subunit; GAP45, Glideosome-Associated Protein 45; CKIIβ, Casein kinase II beta subunit; ANP32, IPP2A-1/ANP32-like protein; SET, IPP2A-2/SET-like protein; SNAP, alpha-soluble NSF attachment protein; PP2C, putative protein phosphatase 2C; ARM1, armadillo repeat-containing protein; MLC1 myosin light chain 1, RAB5B, GTP-binding protein RAB5B; Hypo, hypothetical phosphoprotein.