35s easytag mix

For ³⁵S metabolic labeling, confluent HFF cells in a T75 flask were infected with 1 × 10⁷ tachyzoites and incubated for 16 to 20 h. The infected cells were then incubated in methionine/cysteine-free DMEM (GIBCO) containing 1% (vol/vol) FBS for 1 h, followed by 24 h in DMEM containing 500 μCi ³⁵S-Easytag mix (Perkin Elmer). Infected cells were detached from the flask using a cell scraper, washed twice with ice-cold PBS, and lysed in FLAG lysis buffer for anti-FLAG immunoprecipitation or TX-100 lysis buffer (1%, vol/vol, TX-100, 50 mM Tris HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1:200 [vol/vol] protease inhibitors) for anti-Ty and anti-GAP45 immunoprecipitations. Immunoprecipitation was performed as described above except that, after incubation with primary antibody, protein A-Sepharose (Invitrogen) was added and incubated for 1 h with gentle agitation at 4°C to collect the immune complexes. After three washes with either FLAG wash buffer or TX-100 IP wash buffer (1%, vol/vol, TX-100, 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, and 1:500 protease inhibitors), bound proteins were eluted in SDS-PAGE sample buffer by boiling at 100°C for 5 min. Samples were then resolved by SDS-PAGE and transferred to PVDF membranes for phosphorimaging and immunoblotting.