Toxoplasma Infection Induces ER Stress Pathway

Cells were infected with Toxoplasma for 2 h, washed with PBS, and then cultured in DMEM for the desired times. The infected cells were harvested in radioimmunoprecipitation assay (RIPA) buffer solution supplemented with cOmplete, EDTA-free protease inhibitor cocktail (Roche). Protein quantification was performed using Bradford reagent (Sigma-Aldrich). Equal amounts of protein lysates were separated by SDS-PAGE, and proteins were transferred to nitrocellulose filters. Immunoblot analyses were done using primary antibodies against IRE1 (Abcam; ab37073), XBP1s (Cell Signaling; number D2C1F), ATF6 (30 (link)), GAPDH (Abcam; ab9485), and PERK (Cell Signaling; number 3192), followed by Amersham ECL horseradish peroxidase (HRP)-conjugated secondary antibody. These antibodies and additional reagents used in the study are listed in Table S2. Proteins in the immunoblots were visualized using FluorChem M (multiplex fluorescence; Protein Simple). Immunoblot analyses were carried out for three independent experiments.