Ab37073

Cells or fresh tissues were lysed in RIPA lysis buffer with mixture of protease inhibitors (Beyotime #ST506) and PhosSTOP (Roche #04906845001). 30 μg total proteins were subjected to 12% SDS–polyacrylamide gel. After electrophoresis, the proteins were transferred to PVDF membranes, which were then blocked with 5% milk for 2 hours. The membranes were then probed with primary antibody for IRE1 (Abcam # ab37073), CHOP (Cell Signaling Technology # 2895), GAPDH (Santa Cruz #sc32233), GRP78 (Abcam # ab32618), p-eIF2α (Cell Signaling Technology # 9721), eIF2α (Cell Signaling Technology # 3597), DUOX1 (Abcam #ab78919), p-PERK (Cell Signaling Technology #3179), PERK (Cell Signaling Technology #3192) at 4°C overnight, and then the membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (Santa Cruz # sc-2030) for 1.5 hours and finally washed and visualized using Chemiluminescent ECL reagent (Beyotime # P0018).

The mouse cDNA sequence of filamin A from MEFs was amplified and cloned into pcDNA3-myc plasmid (Addgene). The resulting plasmid, pcDNA3-myc-FLNA, was transiently transfected in MEFs, and then the transfected cells were infected with Toxoplasma for 18 h (MOI, 3). Cell lysates were prepared using IP-lysis solution (0.5% NP-40, 250 mM NaCl, 30 mM Tris, 0.5% glycerol [pH 7.4], 250 mM phenylmethylsulfonyl fluoride [PMSF] supplemented with cOmplete, EDTA-free protease inhibitor cocktail [Roche]). To immunoprecipitate Myc-tagged filamin A (Myc-filamin A), equal amounts of protein lysates were incubated with IgG magnetic beads (Pierce) for 2 h and then mixed with anti-Myc magnetic beads (Pierce) overnight at 4°C with rotation. Proteins bound to the beads were subsequently washed four times with IP-lysis solution at 4°C and then once with IP-lysis solution supplemented with 500 mM NaCl. Protein complexes were eluted at 95°C for 5 min in loading buffer solution and then separated by SDS-PAGE, followed by immunoblot analyses using specific antibodies to IRE1 (Abcam; ab37073) or Myc (Cell Signaling; no. 2276).

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.