FIG 5.
Transfectants expressing HA-tagged CDPK1 proteins. (A) Western blot of parasites overexpressing WT and the indicated mutant CDPK1-HA proteins. Endogenous CDPK1 was also expressed. Lysates bearing the specified number of parasites (in millions) were separated by SDS-PAGE and blotted with rabbit anti-CDPK1 and rabbit anti-MIC10 (a gift of Vern Carruthers) (37). MIC10 was used as a loading control on the same gel and indicates that the H201Q mutant lanes were somewhat overloaded. Mouse anti-HA.11 (clone 16B12; Covance) was used to demonstrate that the upper band revealed with anti-CDPK1 in each lane represents the HA-tagged recombinant protein (not shown). Lane M, molecular markers (in kilodaltons). (B) RM-1-132 EC50s of T. gondii expressing CDPK1 mutant proteins. The clones expressing HA-tagged proteins also expressed endogenous CDPK1. Data were collected in triplicate within each assay, and assays were repeated four or more times [except for the assay with CDPK1(H201Q)-HA, which was done twice]. The bars represent the standard error of the mean.