Nbt and bcip

Tachyzoites were collected, filtered, counted and lysed by 6 cycles of rapid freezing/defreeze in hypotonic buffer, and boiled with LB for 5 minutes. 0.5 to 1×10⁷ parasites were loaded per well and resolved by 15% SDS-PAGE. Proteins were transferred to PVDF membrane for 1h at 100V. Western blot was then performed as described [90 (link)]. The primary antibodies: αrH2B.Z [22 (link)], αrH2A.Z [23 (link)], αrROP5 (rabbit) and αrROP18 (rabbit) were used at 1/5000, whereas α-c-Myc (rabbit, abCam ab9106) was used at 1/2000 and αrSag1 [87 (link)] 1/200 for 1 h at room temperature. αH3ac (rabbit, Millipore, 06–599B) 1/200, was incubated overnight. Appropriate secondary antibodies were used: phosphatase alkaline-conjugated goat anti-mouse or anti-rabbit (Sigma) along with the NBT and BCIP (Promega) detection system.

Seven dpg tir1-1 35S:TIR1-Myc plants were transferred to liquid ATS medium supplemented with 200 mM NaCl for different times. After treatments, plants were homogenized in ice-cold buffer [50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) pH 7.5, 200 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) Tween-20], containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and complete protease inhibitor cocktail (Roche) and centrifuged twice at 10,000 g at 4°C for 15 min. Equal amounts of protein were loaded onto SDS-PAGE and blotted onto the nitrocellulose membrane. Membranes were incubated with anti-c Myc antibody (Sigma) and goat anti-mouse alkaline phosphatase conjugate (Sigma) was used as secondary antibody. Then, Myc detection was visualized with NBT and BCIP (Promega). Densitometry analysis of immunoblots was performed using Matrox Inspector 2.2 software (Matrox Electronics Systems, Ltd).