Anti his6 hrp

N. benthamiana leaf discs were ground in liquid nitrogen and proteins were extracted in Laemmli buffer 2X. Yeast protein were extracted after a lysis step in 0.1 M NaOH during 20 min at room temperature followed by protein extraction in Laemmli buffer 1X. Immunodetection of proteins were performed by loading the samples on precast SDS-PAGE gels (4-15%, Bio-Rad). Proteins were transferred on nitrocellulose membrane using the Trans-Blot Turbo transfer system (Bio-Rad). The nitrocellulose membranes were blocked with 1X Tris-buffered Saline with Tween20 (TBS-T) solution (137 mM NaCl, 0.1% Tween-20, 3% Milk). Proteins transferred on nitrocellulose membranes were stained in Ponceau S staining solution (0.5% Ponceau S (w/v), 1% acetic acid). Immuno-detection of the protein of interest were performed with the following antibodies : anti-GFP (1:3000, mouse mAb (clones 7.1 and 13.1), Sigma), anti-HA-HRP (1:5000, Rat mAb (clone 3F10), Sigma), anti-Gal4-AD (1:5000, mouse mAb, Takara), Anti-Ac-K (1:2000, mouse mAb Ac-K-103, Cell signaling), anti-His6-HRP (1:50000, mouse mAb (clone His-2), Roche), anti-luciferase (1:10000, L0159, Sigma), anti-rabbit IgG-HRP (1:10000, Cell signaling) and polyclonal goat anti-mouse-Hrp (1:10000, Bio-Rad).