Goat anti mouse or anti rabbit secondary antibody conjugated to horseradish peroxidase

Total proteins were extracted from N. benthamiana leaves as described previously [66] (link). Immunoblotting was performed with primary mouse monoclonal or rabbit polyclonal antibodies, followed by goat anti-mouse or anti-rabbit secondary antibody conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA). The GFP monoclonal antibody was obtained from Hua An Company, China, and the monoclonal antibodies against PVX CP, CMV CP and βC1 were generated in house. The rabbit polyclonal antibody against CMV 2b was a generous gift from Dr. Huishan Guo. Blotted membranes were washed thoroughly and visualized using chemiluminescence according to the manufacturer's manual (GE Healthcare). Nbrgs-CaM antibody was produced in rabbit with recombinant protein expressed in E. coli. In brief, we expressed Nbrgs-CaM in E. coli cells as a 6× Histidine and Maltose binding protein (MBP) fusion. The Nbrgs-CaM-MBP-His fusion protein was purified over a nickle column (Merck, Darmstadt, Germany) and eluted with a buffer containing 200 mM imidazole according to the manufacturer's manual. The purified protein was used to immunize rabbits for polyclonal antiserum. The Nbrgs-CaM-specific immunoglubin was given an additional purification step on an affinity column filled with Cyanogen bromide-activated agarose beads conjugated with the Nbrgs-CaM-MBP-His fusion proteins.

Total protein was extracted from infiltrated leaf patches as described previously [67 (link)]. Immunoblotting was performed with primary mouse monoclonal or rabbit polyclonal antibodies, followed by goat anti-mouse or anti-rabbit secondary antibody conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA, USA). The GFP polyclonal antibody was obtained from Abcam (Massachusetts, US), and the Myc monoclonal antibody was obtained from Sigma (Los Angeles, CA, USA). Blotted membranes were washed thoroughly and visualized using chemiluminescence according to the manufacturer’s protocol (ECL; GE Healthcare).